Isolated human secreted proteins, nucleic acid molecules encoding human secreted proteins and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the secreted peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the secreted peptides, and methods of identifying modulators of the secreted peptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of secreted proteins thatare related to the fibroblast growth factor receptor 2 secretedsubfamily, recombinant DNA molecules, and protein production. Thepresent invention specifically provides novel peptides and proteins thateffect protein phosphorylation and nucleic acid molecules encoding suchpeptide and protein molecules, all of which are useful in thedevelopment of human therapeutics and diagnostic compositions andmethods.

BACKGROUND OF THE INVENTION

[0002] Secreted Proteins

[0003] Many human proteins serve as pharmaceutically active compounds.Several classes of human proteins that serve as such active compoundsinclude hormones, cytokines, cell growth factors, and celldifferentiation factors. Most proteins that can be used as apharmaceutically active compound fall within the family of secretedproteins. It is, therefore, important in developing new pharmaceuticalcompounds to identify secreted proteins that can be tested for activityin a variety of animal models. The present invention advances the stateof the art by providing many novel human secreted proteins.

[0004] Secreted proteins are generally produced within cells at roughendoplasmic reticulum, are then exported to the golgi complex, and thenmove to secretory vesicles or granules, where they are secreted to theexterior of the cell via exocytosis.

[0005] Secreted proteins are particularly useful as diagnostic markers.Many secreted proteins are found, and can easily be measured, in serum.For example, a ‘signal sequence trap’ technique can often be utilizedbecause many secreted proteins, such as certain secretory breast cancerproteins, contain a molecular signal sequence for cellular exportAdditionally, antibodies against particular secreted serum proteins canserve as potential diagnostic agents, such as for diagnosing cancer.

[0006] Secreted proteins play a critical role in a wide array ofimportant biological processes in humans and have numerous utilities;several illustrative examples are discussed herein. For example,fibroblast secreted proteins participate in extracellular matrixformation. Extracellular matrix affects growth factor action, celladhesion, and cell growth. Structural and quantitative characteristicsof fibroblast secreted proteins are modified during the course ofcellular aging and such aging related modifications may lead toincreased inhibition of cell adhesion, inhibited cell stimulation bygrowth factors, and inhibited cell proliferative ability (Eleftheriou etal., Mutat Res 1991 March-November;256(2-6):127-38).

[0007] The secreted form of amyloid beta/A4 protein precursor (APP)functions as a growth and/or differentiation factor. The secreted formof APP can stimulate neurite extension of cultured neuroblastoma cells,presumably through binding to a cell surface receptor and therebytriggering intracellular transduction mechanisms. (Roch et al., Ann N YAcad Sci 1993 September 24;695:149-57). Secreted APPs modulate neuronalexcitability, counteract effects of glutamate on growth cone behaviors,and increase synaptic complexity. The prominent effects of secreted APPson synaptogenesis and neuronal survival suggest that secreted APPs playa major role in the process of natural cell death and, furthermore, mayplay a role in the development of a wide variety of neurologicaldisorders, such as stroke, epilepsy, and Alzheimer's disease (Mattson etal., Perspect Dev Neurobiol 1998; 5(4):337-52).

[0008] Breast cancer cells secrete a 52K estrogen-regulated protein (seeRochefort et al., Ann N YAcad Sci 1986;464:190-201). This secretedprotein is therefore usefull in breast cancer diagnosis.

[0009] Two secreted proteins released by platelets, platelet factor 4(PF4) and beta-thromboglobulin (betaTG), are accurate indicators ofplatelet involvement in hemostasis and thrombosis and assays thatmeasure these secreted proteins are useful for studying the pathogenesisand course of thromboembolic disorders (Kaplan, Adv Exp Med Biol1978;102:105-19).

[0010] Vascular endothelial growth factor (VEGF) is another example of anaturally secreted protein VEGF binds to cell-surface heparan sulfates,is generated by hypoxic endothelial cells, reduces apoptosis, and bindsto high-affinity receptors that are up-regulated by hypoxia (Asahara etal., Semin Interv Cardiol 1996 September;1(3):225-32).

[0011] Many critical components of the immune system are secretedproteins, such as antibodies, and many important functions of the immunesystem are dependent upon the action of secreted proteins. For example,Saxon et al., Biochem Soc Trans 1997 May;25(2):383-7, discusses secretedIgE proteins.

[0012] For a further review of secreted proteins, see Nilsen-Hamilton etal., Cell Biol Int Rep 1982 September;6(9):815-36.

[0013] Fibroblast Growth Factor Receptor 2

[0014] The secreted proteins of this invention are a novel form of thecirculating soluble fibroblast growth factor receptor 2, a receptortyrosine kinase. Fibroblast growth factor receptor 2 is also known asK-sam II. The K-sam gene was first identified as a gene amplified in thestomach cancer cell line KATO-III. The K-sam gene expresses multiplesizes of mRNAs in brain tissue, the immature teratoma cell line NCC-IT,and KATO-III. RNA blot analyses with a variety of K-sam probes indicatethat there are at least four classes of K-sam mRNAs. Experimentalevidence indicates that the gene products of these classes are: (1) areceptor tyrosine kinase that belongs to the heparin-binding growthfactor receptor, or fibroblast growth factor receptor, gene family; (2)a full-length transmembrane receptor; (3) a secreted receptor with atyrosine kinase domain; and (4) a secreted receptor without a tyrosinekinase domain. For more information on K-sam proteins, see Katoh, etal., Proc Natl Acad Sci USA 1992 April 1;89(7):29604.

[0015] The novel form of this circulating soluble fibroblast growthfactor receptor 2 exhibits a high degree of identity with a known FGFreceptor 2, with an additional two amino acid residues at itsC-terminal. The stop codon at the end of the open reading frame and apolyadenylation signal located 19 bp upstream from the 3′end are presentin the genomic sequence, indicating that the 3′end is intact Theproteins of this invention comprise a signal peptide (residues 1-40),and a first Ig-like domain (residues 74-128), similar to K-Sam IV, thesecreted FGF receptor 2 without a tyrosine kinase domain.

[0016] Like other secreted receptors, the novel proteins of thisinvention may act as carrier proteins. They may modulate the response oftarget cells to ligands, or alternatively may transduce signals asreceptors for the secreted interleulin 6 receptor.

[0017] Secreted proteins, particularly members of the fibroblast growthfactor receptor 2 secreted protein subfamily, are a major target fordrug action and development. Accordingly, it is valuable to the field ofpharmaceutical development to identify and characterize previouslyunknown members of this subfamily of secreted proteins. The presentinvention advances the state of the art by providing previouslyunidentified human secreted proteins that have homology to members ofthe fibroblast growth factor receptor 2 secreted protein subfamily.

SUMMARY OF THE INVENTION

[0018] The present invention is based in part on the identification ofamino acid sequences of human secreted peptides and proteins that arerelated to the fibroblast growth factor receptor 2 secreted proteinsubfamily, as well as allelic variants and other mammalian orthologsthereof. These unique peptide sequences, and nucleic acid sequences thatencode these peptides, can be used as models for the development ofhuman therapeutic targets, aid in the identification of therapeuticproteins, and serve as targets for the development of human therapeuticagents that modulate secreted protein activity in cells and tissues thatexpress the secreted protein. Experimental data as provided in FIG. 1indicates expression in the testis, nervous tissue, fetal lung, brainanaplastic oligodendroglioma, lung carcinoid tissue, soft tissueleiomyosarcoma, ovary tumor tissue, and germ cell tumor tissue.

DESCRIPTION OF THE FIGURE SHEETS

[0019]FIG. 1 provides the nucleotide sequence of a cDNA molecule ortranscript sequence that encodes the secreted protein of the presentinvention. (SEQ ID NO:1) In addition, structure and functionalinformation is provided, such as ATG start, stop and tissuedistribution, where available, that allows one to readily determinespecific uses of inventions based on this molecular sequence.Experimental data as provided in FIG. 1 indicates expression in thetestis, nervous tissue, fetal lung, brain anaplastic oligodendroglioma,lung carcinoid tissue, soft tissue leiomyosarcoma, ovary tumor tissue,and germ cell tumor tissue.

[0020]FIG. 2 provides the predicted amino acid sequence of the secretedprotein of the present invention. (SEQ ID NO:2) In addition structureand functional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

[0021]FIG. 3 provides genomic sequences that span the gene encoding thesecreted protein of the present invention. (SEQ ID NO:3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. As illustrated in FIG. 3, SNPs were identified at 40 differentnucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION

[0022] General Description

[0023] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a secreted protein or part of asecreted protein and are related to the fibroblast growth factorreceptor 2 secreted protein subfamily. Utilizing these sequences,additional genomic sequences were assembled and transcript and/or cDNAsequences were isolated and characterized. Based on this analysis, thepresent invention provides amino acid sequences of human secretedpeptides and proteins that are related to the fibroblast growth factorreceptor 2 secreted protein subfamily, nucleic acid sequences in theform of transcript sequences, cDNA sequences and/or genomic sequencesthat encode these secreted peptides and proteins, nucleic acid variation(allelic information), tissue distribution of expression, andinformation about the closest art known protein/peptide/domain that hasstructural or sequence homology to the secreted protein of the presentinvention.

[0024] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known secreted proteins of thefibroblast growth factor receptor 2 secreted protein subfamily and theexpression pattern observed. Experimental data as provided in FIG. 1indicates expression in the testis, nervous tissue, fetal lung, brainanaplastic oligodendroglioma, lung carcinoid tissue, soft tissueleiomyosarcoma, ovary tumor tissue, and germ cell tumor tissue. The arthas clearly established the commercial importance of members of thisfamily of proteins and proteins that have expression patterns similar tothat of the present gene. Some of the more specific features of thepeptides of the present invention, and the uses thereof, are describedherein, particularly in the Background of the Invention and in theannotation provided in the Figures, and/or are known within the art foreach of the known fibroblast growth factor receptor 2 family orsubfamily of secreted proteins.

SPECIFIC EMBODIMENTS

[0025] Peptide Molecules

[0026] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thesecreted protein family of proteins and are related to the fibroblastgrowth factor receptor 2 secreted protein subfamily (protein sequencesare provided in FIG. 2, transcript/cDNA sequences are provided in FIG. 1and genomic sequences are provided in FIG. 3). The peptide sequencesprovided in FIG. 2, as well as the obvious variants described herein,particularly allelic variants as identified herein and using theinformation in FIG. 3, will be referred herein as the secreted peptidesof the present invention, secreted peptides, or peptides/proteins of thepresent invention.

[0027] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprise the aminoacid sequences of the secreted peptides disclosed in the FIG. 2,(encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNAor FIG. 3, genomic sequence), as well as all obvious variants of thesepeptides that are within the art to make and use. Some of these variantsare described in detail below.

[0028] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0029] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0030] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thesecreted peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0031] The isolated secreted peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inthe testis, nervous tissue, fetal lung, brain anaplasticoligodendroglioma, lung carcinoid tissue, soft tissue leiomyosarcoma,ovary tumor tissue, and germ cell tumor tissue. For example, a nucleicacid molecule encoding the secreted peptide is cloned into an expressionvector, the expression vector introduced into a host cell and theprotein expressed in the host cell. The protein can then be isolatedfrom the cells by an appropriate purification scheme using standardprotein purification techniques. Many of these techniques are describedin detail below.

[0032] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG.3 (SEQ ID NO:3). The amino acid sequence of such a protein is providedin FIG. 2. A protein consists of an amino acid sequence when the aminoacid sequence is the final amino acid sequence of the protein.

[0033] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ IDNO:2), for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequencesprovided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of anamino acid sequence when such an amino acid sequence is present withonly a few additional amino acid residues, for example from about 1 toabout 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

[0034] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the secreted peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

[0035] The secreted peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a secreted peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the secreted peptide. “Operatively linked”indicates that the secreted peptide and the heterologous protein arefused in-frame. The heterologous protein can be fused to the N-termiinusor C-terminus of the secreted peptide.

[0036] In some uses, the fusion protein does not affect the activity ofthe secreted peptideper se. For example, the fusion protein can include,but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant secreted peptide. In certain host cells (e.g., mammalianhost cells), expression and/or secretion of a protein can be increasedby using a heterologous signal sequence.

[0037] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A secreted peptide-encoding nucleicacid can be cloned into such an expression vector such that the fusionmoiety is linked in-frame to the secreted peptide.

[0038] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0039] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the secreted peptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

[0040] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of the length of a reference sequence is aligned forcomparison purposes. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0041] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

[0042] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0043] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the secreted peptides of the present invention as well asbeing encoded by the same genetic locus as the secreted peptide providedherein.

[0044] Allelic variants of a secreted peptide can readily be identifiedas being a human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the secreted peptide as wellas being encoded by the same genetic locus as the secreted peptideprovided herein. Genetic locus can readily be determined based on thegenomic information provided in FIG. 3, such as the genomic sequencemapped to the reference human. As used herein, two proteins (or a regionof the proteins) have significant homology when the amino acid sequencesare typically at least about 70-80%, 80-90%, and more typically at leastabout 90-95% or more homologous. A significantly homologous amino acidsequence, according to the present invention, will be encoded by anucleic acid sequence that will hybridize to a secreted peptide encodingnucleic acid molecule under stringent conditions as more fully describedbelow.

[0045]FIG. 3 provides information on SNPs that have been found in thegene encoding the transporter protein of the present invention. SNPswere identified at 40 different nucleotide positions, including anon-synonymous coding SNP at position 124. Changes in the amino acidsequence caused by these SNPs is indicated in FIG. 3 and can readily bedetermined using the universal genetic code and the protein sequenceprovided in FIG. 2 as a reference. Some of these SNPs may affect genetranscription. Positioning of each SNP in an exon, intron, or outsidethe ORF can readily be determined using the DNA position given for eachSNP and the start/stop, exon, and intron genomic coordinates given inFIG. 3.

[0046] Paralogs of a secreted peptide can readily be identified ashaving some degree of significant D sequence homology/identity to atleast a portion of the secreted peptide, as being encoded by a gene fromhumans, and as having similar activity or function. Two proteins willtypically be considered paralogs when the amino acid sequences aretypically at least about 60% or greater, and more typically at leastabout 70% or greater homology through a given region or domain. Suchparalogs will be encoded by a nucleic acid sequence that will hybridizeto a secreted peptide encoding nucleic acid molecule under moderate tostringent conditions as more fully described below.

[0047] Orthologs of a secreted peptide can readily be identified ashaving some degree of significant sequence homology/identity to at leasta portion of the secreted peptide as well as being encoded by a genefrom another organism. Preferred orthologs will be isolated frommammals, preferably primates, for the development of human therapeutictargets and agents. Such orthologs will be encoded by a nucleic acidsequence that will hybridize to a secreted peptide encoding nucleic acidmolecule under moderate to stringent conditions, as more fully describedbelow, depending on the degree of relatedness of the two organismsyielding the proteins.

[0048] Non-naturally occurring variants of the secreted peptides of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the secreted peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a secreted peptide by another amino acid of likecharacteristics. Typically seen as conservative substitutions are thereplacements, one for another, among the aliphatic amino acids Ala, Val,Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchangeof the acidic residues Asp and Glu; substitution between the amideresidues Asn and Gln; exchange of the basic residues Lys and Arg; andreplacements among the aromatic residues Phe and Tyr. Guidanceconcerning which amino acid changes are likely to be phenotypicallysilent are found in Bowie et al., Science 247:1306-1310 (1990).

[0049] Variant secreted peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind substrate,ability to phosphorylate substrate, ability to mediate signaling, etc.Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

[0050] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0051] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989>), particularly using the results provided in FIG. 2. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as secreted protein activity or in assays such as an invitro proliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

[0052] The present invention further provides fragments of the secretedpeptides, in addition to proteins and peptides that comprise and consistof such fragments, particularly those comprising the residues identifiedin FIG. 2. The fragments to which the invention pertains, however, arenot to be construed as encompassing fragments that may be disclosedpublicly prior to the present invention.

[0053] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a secreted peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the secreted peptide or could be chosen forthe ability to perform a function, e.g. bind a substrate or act as animmunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe secreted peptide, e.g., active site or a substrate-binding domain.Further, possible fragments include, but are not limited to, domain ormotif containing fragments, soluble peptide fragments, and fragmentscontaining immunogenic structures. Predicted domains and functionalsites are readily identifiable by computer programs well known andreadily available to those of skill in the art (e.g., PROSITE analysis).The results of one such analysis are provided in FIG. 2.

[0054] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally insecreted peptides are described in basic texts, detailed monographs, andthe research literature, and they are well known to those of skill inthe art (some of these features are identified in FIG. 2).

[0055] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0056] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

[0057] Accordingly, the secreted peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature secreted peptide is fused withanother compound, such as a compound to increase the half-life of thesecreted peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature secreted peptide, such asa leader or secretory sequence or a sequence for purification of themature secreted peptide or a pro-protein sequence.

[0058] Protein/Peptide Uses

[0059] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a secretedprotein-effector protein interaction or secreted protein-ligandinteraction), the protein can be used to identify the bindingpartner/ligand so as to develop a system to identify inhibitors of thebinding interaction. Any or all of these uses are capable of beingdeveloped into reagent grade or kit format for commercialization ascommercial products.

[0060] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0061] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well- as theclass/action of the protein. For example, secreted proteins isolatedfrom humans and their human/mammalian orthologs serve as targets foridentifying agents for use in mammalian therapeutic applications, e.g. ahuman drug, particularly in modulating a biological or pathologicalresponse in a cell or tissue that expresses the secreted protein.Experimental data as provided in FIG. 1 indicates that secreted proteinsof the present invention are expressed in the testis, nervous tissue,fetal lung, brain anaplastic oligodendroglioma, lung carcinoid tissue,soft tissue leiomyosarcoma, ovary tumor tissue, and germ cell tumortissue. Specifically, a virtual northern blot shows expression in thetestis, nervous tissue, fetal lung, brain anaplastic oligodendroglioma,lung carcinoid tissue, soft tissue leiomyosarcoma, ovary tumor tissue,and germ cell tumor tissue. A large percentage of pharmaceutical agentsare being developed that modulate the activity of secreted proteins,particularly members of the fibroblast growth factor receptor 2subfamily (see Background of the Invention). The structural andfunctional information provided in the Background and Figures providespecific and substantial uses for the molecules of the presentinvention, particularly in combination with the expression informationprovided in FIG. 1. Experimental data as provided in FIG. 1 indicatesexpression in the testis, nervous tissue, fetal lung, brain anaplasticoligodendroglioma, lung carcinoid tissue, soft tissue leiomyosarcoma,ovary tumor tissue, and germ cell tumor tissue. Such uses can readily bedetermined using the information provided herein, that which is known inthe art, and routine experimentation.

[0062] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to secreted proteins that arerelated to members of the fibroblast growth factor receptor 2 subfamily.Such assays involve any of the known secreted protein functions oractivities or properties useful for diagnosis and treatment of secretedprotein-related conditions that are specific for the subfamily ofsecreted proteins that the one of the present invention belongs to,particularly in cells and tissues that express the secreted protein.Experimental data as provided in FIG. 1 indicates that secreted proteinsof the present invention are expressed in the testis, nervous tissue,fetal lung, brain anaplastic oligodendroglioma, lung carcinoid tissue,soft tissue leiomyosarcoma, ovary tumor tissue, and germ cell tumortissue. Specifically, a virtual northern blot shows expression in thetestis, nervous tissue, fetal lung, brain anaplastic oligodendroglioma,lung carcinoid tissue, soft tissue leiomyosarcoma, ovary tumor tissue,and germ cell tumor tissue.

[0063] The proteins of the present invention are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the secreted protein,as a biopsy or expanded in cell culture. Experimental data as providedin FIG. 1 indicates expression in the testis, nervous tissue, fetallung, brain anaplastic oligodendroglioma, lung carcinoid tissue, softtissue leiomyosarcoma, ovary tumor tissue, and germ cell tumor tissue.In an alternate embodiment, cell-based assays involve recombinant hostcells expressing the secreted protein.

[0064] The polypeptides can be used to identify compounds that modulatesecreted protein activity of the protein in its natural state or analtered form that causes a specific disease or pathology associated withthe secreted protein. Both the secreted proteins of the presentinvention and appropriate variants and fragments can be used inhigh-throughput screens to assay candidate compounds for the ability tobind to the secreted protein. These compounds can be further screenedagainst a functional secreted protein to determine the effect of thecompound on the secreted protein activity. Further, these compounds canbe tested in annual or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the secreted protein to a desireddegree.

[0065] Further, the proteins of the present invention can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the secreted protein and a molecule that normally interacts withthe secreted protein, e.g. a substrate or a component of the signalpathway that the secreted protein normally interacts (for example,another secreted protein). Such assays typically include the steps ofcombining the secreted protein with a candidate compound underconditions that allow the secreted protein, or fragment, to interactwith the target molecule, and to detect the formation of a complexbetween the protein and the target or to detect the biochemicalconsequence of the interaction with the secreted protein and the target.

[0066] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al. Nature 354:82-84(1991);Houghten et al., Nature 354:84-86(1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0067] One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantsecreted proteins or appropriate fragments containing mutations thataffect secreted protein function and thus compete for substrate.Accordingly, a fragment that competes for substrate, for example with ahigher affinity, or a fragment that binds substrate but does not allowrelease, is encompassed by the invention.

[0068] Any of the biological or biochemical functions mediated by thesecreted protein can be used as an endpoint assay. These include all ofthe biochemical or biochemical/biological events described herein, inthe references cited herein, incorporated by reference for theseendpoint assay targets, and other functions known to those of ordinaryskill in the art or that can be readily identified using the informationprovided in the Figures, particularly FIG. 2. Specifically, a biologicalfunction of a cell or tissues that expresses the secreted protein can beassayed. Experimental data as provided in FIG. 1 indicates that secretedproteins of the present invention are expressed in the testis, nervoustissue, fetal lung, brain anaplastic oligodendroglioma, lung carcinoidtissue, soft tissue leiomyosarcoma, ovary tumor tissue, and germ celltumor tissue. Specifically, a virtual northern blot shows expression inthe testis, nervous tissue, fetal lung, brain anaplasticoligodendroglioma, lung carcinoid tissue, soft tissue leiomyosarcoma,ovary tumor tissue, and germ cell tumor tissue.

[0069] Binding and/or activating compounds can also be screened by usingchimeric secreted proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native secreted protein. Accordingly, a different setof signal transduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the secreted protein is derived.

[0070] The proteins of the present invention are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the secreted protein (e.g. binding partners and/orligands). Thus, a compound is exposed to a secreted protein polypeptideunder conditions that allow the compound to bind or to otherwiseinteract with the polypeptide. Soluble secreted protein polypeptide isalso added to the mixture. If the test compound interacts with thesoluble secreted protein polypeptide, it decreases the amount of complexformed or activity from the secreted protein target. This type of assayis particularly useful in cases in which compounds are sought thatinteract with specific regions of the secreted protein. Thus, thesoluble polypeptide that competes with the target secreted proteinregion is designed to contain peptide sequences corresponding to theregion of interest.

[0071] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the secreted protein, or fragment, or itstarget molecule to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay.

[0072] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of secreted protein-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a secreted protein-binding protein and a candidate compound areincubated in the secreted protein-presenting wells and the amount ofcomplex trapped in the well can be quantitated. Methods for detectingsuch complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the secreted protein target molecule, or whichare reactive with secreted protein and compete with the target molecule,as well as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the target molecule.

[0073] Agents that modulate one of the secreted proteins of the presentinvention can be identified using one or more of the above assays, aloneor in combination. It is generally preferable to use a cell-based orcell free system first and then confirm activity in an animal or othermodel system. Such model systems are well known in the art and canreadily be employed in this context.

[0074] Modulators of secreted protein activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the secreted protein pathway, by treating cells ortissues that express the secreted protein. Experimental data as providedin FIG. 1 indicates expression in the testis, nervous tissue, fetallung, brain anaplastic oligodendroglioma, lung carcinoid tissue, softtissue leiomyosarcoma, ovary tumor tissue, and germ cell tumor tissue.These methods of treatment include the steps of administering amodulator of secreted protein activity in a pharmaceutical compositionto a subject in need of such treatment, the modulator being identifiedas described herein.

[0075] In yet another aspect of the invention, the secreted proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the secreted protein and are involved insecreted protein activity.

[0076] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a secreted proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming a secretedprotein-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the secreted protein.

[0077] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a secreted protein-modulating agent, anantisense secreted protein nucleic acid molecule, a secretedprotein-specific antibody, or a secreted protein-binding partner) can beused in an animal or other model to determine the efficacy, toxicity, orside effects of treatment with such an agent. Alternatively, an agentidentified as described herein can be used in an animal or other modelto determine the mechanism of action of such an agent. Furthermore, thisinvention pertains to uses of novel agents identified by theabove-described screening assays for treatments as described herein.

[0078] The secreted proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of the protein (or encoding mRNA) in acell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in the testis, nervous tissue, fetal lung, brainanaplastic oligodendroglioma, lung carcinoid tissue, soft tissueleiomyosarcoma, ovary tumor tissue, and germ cell tumor tissue. Themethod involves contacting a biological sample with a compound capableof interacting with the secreted protein such that the interaction canbe detected. Such an assay can be provided in a single detection formator a multi-detection format such as an antibody chip array.

[0079] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject The peptides of thepresent invention also provide targets for diagnosing active proteinactivity, disease, or predisposition to disease, in a patient having avariant peptide, particularly activities and conditions that are knownfor other members of the family of proteins to which the present onebelongs. Thus, the peptide can be isolated from a biological sample andassayed for the presence of a genetic mutation that results in aberrantpeptide. This includes amino acid substitution, deletion, insertion,rearrangement, (as the result of aberrant splicing events), andinappropriate post-translational modification. Analytic methods includealtered electrophoretic mobility, altered tryptic peptide digest,altered secreted protein activity in cell-based or cell-free assay,alteration in substrate or antibody-binding pattern, altered isoelectricpoint, direct amino acid sequencing, and any other of the known assaytechniques useful for detecting mutations in a protein. Such an assaycan be provided in a single detection format or a multi-detection formatsuch as an antibody chip array.

[0080] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody or other types of detection agent. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. Particularly useful are methods that detect the allelicvariant of a peptide expressed in a subject and methods which detectfragments of a peptide in a sample.

[0081] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the secreted protein in which one ormore of the secreted protein functions in one population is differentfrom those in another population. The peptides thus allow a target toascertain a genetic predisposition that can affect treatment modality.Thus, in a ligand-based treatment, polymorphism may give rise to aminoterminal extracellular domains and/or other substrate-binding regionsthat are more or less active in substrate binding, and secreted proteinactivation. Accordingly, substrate dosage would necessarily be modifiedto maximize the therapeutic effect within a given population containinga polymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

[0082] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Experimental data as provided in FIG. 1 indicatesexpression in the testis, nervous tissue, fetal lung, brain anaplasticoligodendroglioma, lung carcinoid tissue, soft tissue leiomyosarcoma,ovary tumor tissue, and germ cell tumor tissue. Accordingly, methods fortreatment include the use of the secreted protein or fragments.

[0083] Antibodies

[0084] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0085] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0086] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0087] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, such as the domainsidentified in FIG. 2, and domain of sequence homology or divergenceamongst the family, such as those that can readily be identified usingprotein alignment methods and as presented in the Figures.

[0088] Antibodies are preferably prepared from regions or discretefragments of the secreted proteins. Antibodies can be prepared from anyregion of the peptide as described herein. However, preferred regionswill include those involved in function/activity and/or secretedprotein/binding partner interaction. FIG. 2 can be used to identifyparticularly important regions while sequence alignment can be used toidentify conserved and unique sequence fragments.

[0089] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0090] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0091] Antibody Uses

[0092] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development Experimental data as provided in FIG. 1 indicatesthat secreted proteins of the present invention are expressed in thetestis, nervous tissue, fetal lung, brain anaplastic oligodendroglioma,lung carcinoid tissue, soft tissue leiomyosarcoma, ovary tumor tissue,and germ cell tumor tissue. Specifically, a virtual northern blot showsexpression in the testis, nervous tissue, fetal lung, brain anaplasticoligodendroglioma, lung carcinoid tissue, soft tissue leiomyosarcoma,ovary tumor tissue, and germ cell tumor tissue. Further, such antibodiescan be used to detect protein in situ, in vitro, or in a cell lysate orsupernatant in order to evaluate the abundance and pattern ofexpression. Also, such antibodies can be used to assess abnormal tissuedistribution or abnormal expression during development or progression ofa biological condition. Antibody detection of circulating fragments ofthe full length protein can be used to identify turnover.

[0093] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. Experimental data as provided in FIG. 1indicates expression in the testis, nervous tissue, fetal lung, brainanaplastic oligodendroglioma, lung carcinoid tissue, soft tissueleiomyosarcoma, ovary tumor tissue, and germ cell tumor tissue. If adisorder is characterized by a specific mutation in the protein,antibodies specific for this mutant protein can be used to assay for thepresence of the specific mutant protein.

[0094] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in thetestis, nervous tissue, fetal lung, brain anaplastic oligodendroglioma,lung carcinoid tissue, soft tissue leiomyosarcoma, ovary tumor tissue,and germ cell tumor tissue. The diagnostic uses can be applied, not onlyin genetic testing, but also in monitoring a treatment modality.Accordingly, where treatment is ultimately aimed at correctingexpression level or the presence of aberrant sequence and aberranttissue distribution or developmental expression, antibodies directedagainst the protein or relevant fragments can be used to monitortherapeutic efficacy.

[0095] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0096] The antibodies are also useful for tissue typing. Experimentaldata as provided in FIG. 1 indicates expression in the testis, nervoustissue, fetal lung, brain anaplastic oligodendroglioma, lung carcinoidtissue, soft tissue leiomyosarcoma, ovary tumor tissue, and germ celltumor tissue. Thus, where a specific protein has been correlated withexpression in a specific tissue, antibodies that are specific for thisprotein can be used to identify a tissue type.

[0097] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the secreted peptide to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

[0098] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nuleic acid arrays and similar methods have been developed forantibody arrays.

[0099] Nucleic Acid Molecules

[0100] The present invention further provides isolated nucleic acidmolecules that encode a secreted peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the secreted peptides of thepresent invention, an allelic variant thereof, or an ortholog or paralogthereof.

[0101] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0102] Moreover, an “isolated” nucleic acid molecule, such as atranscript/cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.However, the nucleic acid molecule can be fused to other coding orregulatory sequences and still be considered isolated.

[0103] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0104] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequencewhen the nucleotide sequence is the complete nucleotide sequence of thenucleic acid molecule.

[0105] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists essentially of anucleotide sequence when such a nucleotide sequence is present with onlya few additional nucleic acid residues in the final nucleic acidmolecule.

[0106] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ IDNO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO:2. A nucleic acid molecule comprises a nucleotide sequence whenthe nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can comprisesseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

[0107] In FIGS. 1 and 3, both coding and non-coding sequences areprovided. Because of the source of the present invention, humans genomicsequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleicacid molecules in the Figures will contain genomic intronic sequences,5′ and 3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

[0108] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0109] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the secreted peptidealone, the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

[0110] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0111] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the secreted proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0112] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents. A promoter can readily be identified as being 5′to the ATG start site in the genomic sequence provided in FIG. 3.

[0113] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0114] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0115] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art As described in the Peptide Section, thesevariants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene.

[0116]FIG. 3 provides information on SNPs that have been found in thegene encoding the transporter protein of the present invention. SNPswere identified at 40 different nucleotide positions, including anon-synonymous coding SNP at position 124. Changes in the amino acidsequence caused by these SNPs is indicated in FIG. 3 and can readily bedetermined using the universal genetic code and the protein sequenceprovided in FIG. 2 as a reference. Some of these SNPs may affect genetranscription. Positioning of each SNP in an exon, intron, or outsidethe ORF can readily be determined using the DNA position given for eachSNP and the start/stop, exon, and intron genomic coordinates given inFIG. 3.

[0117] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45 C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65 C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

[0118] Nucleic Acid Molecule Uses

[0119] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, transcript/cDNA and genomic DNA to isolate full-lengthcDNA and genomic clones encoding the peptide described in FIG. 2 and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in FIG. 2.As illustrated in FIG. 3, SNPs were identified at 40 differentnucleotide positions.

[0120] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from 5′ noncoding regions, the coding region, and 3′noncoding regions. However, as discussed, fragments are not to beconstrued as encompassing fragments disclosed prior to the presentinvention.

[0121] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0122] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0123] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0124] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods.

[0125] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0126] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part of the mRNA produced from thenucleic acid molecules described herein.

[0127] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0128] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0129] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0130] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Experimental data as provided in FIG. 1indicates that secreted proteins of the present invention are expressedin the testis, nervous tissue, fetal lung, brain anaplasticoligodendroglioma, lung carcinoid tissue, soft tissue leiomyosarcoma,ovary tumor tissue, and germ cell tumor tissue. Specifically, a virtualnorthern blot shows expression in the testis, nervous tissue, fetallung, brain anaplastic oligodendroglioma, lung carcinoid tissue, softtissue leiomyosarcoma, ovary tumor tissue, and germ cell tumor tissue.Accordingly, the probes can be used to detect the presence of, or todetermine levels of, a specific nucleic acid molecule in cells, tissues,and in organisms. The nucleic acid whose level is determined can be DNAor RNA. Accordingly, probes corresponding to the peptides describedherein can be used to assess expression and/or gene copy number in agiven cell, tissue, or organism. These uses are relevant for diagnosisof disorders involving an increase or decrease in secreted proteinexpression relative to normal results.

[0131] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA include Southern hybridizations and in situ hybridization.

[0132] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a secreted protein, such as bymeasuring a level of a secreted protein-encoding nucleic acid in asample of cells from a subject e.g., mRNA or genomic DNA, or determiningif a secreted protein gene has been mutated. Experimental data asprovided in FIG. 1 indicates that secreted proteins of the presentinvention are expressed in the testis, nervous tissue, fetal lung, brainanaplastic oligodendroglioma, lung carcinoid tissue, soft tissueleiomyosarcoma, ovary tumor tissue, and germ cell tumor tissue.Specifically, a virtual northern blot shows expression in the testis,nervous tissue, fetal lung, brain anaplastic oligodendroglioma, lungcarcinoid tissue, soft tissue leiomyosarcoma, ovary tumor tissue, andgerm cell tumor tissue.

[0133] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate secreted protein nucleic acidexpression.

[0134] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the secreted protein gene, particularly biological andpathological processes that are mediated by the secreted protein incells and tissues that express it. Experimental data as provided in FIG.1 indicates expression in the testis, nervous tissue, fetal lung, brainanaplastic oligodendroglioma, lung carcinoid tissue, soft tissueleiomyosarcoma, ovary tumor tissue, and germ cell tumor tissue. Themethod typically includes assaying the ability of the compound tomodulate the expression of the secreted protein nucleic acid and thusidentifying a compound that can be used to treat a disordercharacterized by undesired secreted protein nucleic acid expression. Theassays can be performed in cell-based and cell-free systems. Cell-basedassays include cells naturally expressing the secreted protein nucleicacid or recombinant cells genetically engineered to express specificnucleic acid sequences.

[0135] Thus, modulators of secreted protein gene expression can beidentified in a method wherein a cell is contacted with a candidatecompound and the expression of mRNA determined. The level of expressionof secreted protein mRNA in the presence of the candidate compound iscompared to the level of expression of secreted protein mRNA in theabsence of the candidate compound. The candidate compound can then beidentified as a modulator of nucleic acid expression based on thiscomparison and be used, for example to treat a disorder characterized byaberrant nucleic acid expression. When expression of mRNA isstatistically significantly greater in the presence of the candidatecompound than in its absence, the candidate compound is identified as astimulator of nucleic acid expression. When nucleic acid expression isstatistically significantly less in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of nucleic acid expression.

[0136] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate secreted protein nucleic acidexpression in cells and tissues that express the secreted protein.Experimental data as provided in FIG. 1 indicates that secreted proteinsof the present invention are expressed in the testis, nervous tissue,fetal lung, brain anaplastic oligodendroglioma, lung carcinoid tissue,soft tissue leiomyosarcoma, ovary tumor tissue, and germ cell tumortissue. Specifically, a virtual northern blot shows expression in thetestis, nervous tissue, fetal lung, brain anaplastic oligodendroglioma,lung carcinoid tissue, soft tissue leiomyosarcoma, ovary tumor tissue,and germ cell tumor tissue. Modulation includes both up-regulation (i.e.activation or agonization) or down-regulation (suppression orantagonization) or nucleic acid expression.

[0137] Alternatively, a modulator for secreted protein nucleic acidexpression can be a small molecule or drug identified using thescreening assays described herein as long as the drug or small moleculeinhibits the secreted protein nucleic acid expression in the cells andtissues that express the protein. Experimental data as provided in FIG.1 indicates expression in the testis, nervous tissue, fetal lung, brainanaplastic oligodendroglioma, lung carcinoid tissue, soft tissueleiomyosarcoma, ovary tumor tissue, and germ cell tumor tissue.

[0138] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe secreted protein gene in clinical trials or in a treatment regimen.Thus, the gene expression pattern can serve as a barometer for thecontinuing effectiveness of treatment with the compound, particularlywith compounds to which a patient can develop resistance. The geneexpression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound.Accordingly, such monitoring would allow either increased administrationof the compound or the administration of alternative compounds to whichthe patient has not become resistant Similarly, if the level of nucleicacid expression falls below a desirable level, administration of thecompound could be commensurately decreased.

[0139] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in secreted protein nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in secreted protein genesand gene expression products such as mRNA. The nucleic acid moleculescan be used as hybridization probes to detect naturally occurringgenetic mutations in the secreted protein gene and thereby to determinewhether a subject with the mutation is at risk for a disorder caused bythe mutation. Mutations include deletion, addition, or substitution ofone or more nucleotides in the gene, chromosomal rearrangement, such asinversion or transposition, modification of genomic DNA, such asaberrant methylation patterns or changes in gene copy number, such asamplification. Detection of a mutated form of the secreted protein geneassociated with a dysfunction provides a diagnostic tool for an activedisease or susceptibility to disease when the disease results fromoverexpression, underexpression, or altered expression of a secretedprotein.

[0140] Individuals carrying mutations in the secreted protein gene canbe detected at the nucleic acid level by a variety of techniques. FIG. 3provides information on SNPs that have been found in the gene encodingthe transporter protein of the present invention. SNPs were identifiedat 40 different nucleotide positions, including a non-synonymous codingSNP at position 124. Changes in the amino acid sequence caused by theseSNPs is indicated in FIG. 3 and can readily be determined using theuniversal genetic code and the protein sequence provided in FIG. 2 as areference. Some of these SNPs may affect gene transcription. Positioningof each SNP in an exon, intron, or outside the ORF can readily bedetermined using the DNA position given for each SNP and the start/stop,exon, and intron genomic coordinates given in FIG. 3. Genomic DNA can beanalyzed directly or can be amplified by using PCR prior to analysis.RNA or cDNA can be used in the same way. In some uses, detection of themutation involves the use of a probe/primer in a polymerase chainreaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), suchas anchor PCR or RACE PCR, or, alternatively, in a ligation chainreaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080(1988); and Nakazawa et al., PNAS91:360-364 (1994)), the latter of whichcan be particularly useful for detecting point mutations in the gene(see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This methodcan include the steps of collecting a sample of cells from a patient,isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to a gene under conditions such thathybridization and amplification of the gene (if present) occurs, anddetecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

[0141] Alternatively, mutations in a secreted protein gene can bedirectly identified, for example, by alterations in restriction enzymedigestion patterns determined by gel electrophoresis.

[0142] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0143] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method. Furthermore, sequence differences between amutant secreted protein gene and a wild-type gene can be determined bydirect DNA sequencing. A variety of automated sequencing procedures canbe utilized when performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

[0144] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al, Genet.Anal. Tech Appl. 9:73-79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.,Nature 313:495 (1985)). Examples of other techniques for detecting pointmutations include selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

[0145] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the secreted protein gene in an individual in order to selectan appropriate compound or dosage regimen for treatment FIG. 3 providesinformation on SNPs that have been found in the gene encoding thetransporter protein of the present invention. SNPs were identified at 40different nucleotide positions, including a non-synonymous coding SNP atposition 124. Changes in the amino acid sequence caused by these SNPs isindicated in FIG. 3 and can readily be determined using the universalgenetic code and the protein sequence provided in FIG. 2 as a reference.Some of these SNPs may affect gene transcription. Positioning of eachSNP in an exon, intron, or outside the ORF can readily be determinedusing the DNA position given for each SNP and the start/stop, exon, andintron genomic coordinates given in FIG. 3.

[0146] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0147] The nucleic acid molecules are thus useful as antisenseconstructs to control secreted protein gene expression in cells,tissues, and organisms. A DNA antisense nucleic acid molecule isdesigned to be complementary to a region of the gene involved intranscription, preventing transcription and hence production of secretedprotein An antisense RNA or DNA nucleic acid molecule would hybridize tothe mRNA and thus block translation of mRNA into secreted protein.

[0148] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of secreted proteinnucleic acid. Accordingly, these molecules can treat a disordercharacterized by abnormal or undesired secreted protein nucleic acidexpression. This technique involves cleavage by means of ribozymescontaining nucleotide sequences complementary to one or more regions inthe mRNA that attenuate the ability of the mRNA to be translated.Possible regions include coding regions and particularly coding regionscorresponding to the catalytic and other functional activities of thesecreted protein, such as substrate binding.

[0149] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in secreted protein geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desiredsecreted protein to treat the individual.

[0150] The invention also encompasses kits for detecting the presence ofa secreted protein nucleic acid in a biological sample. Experimentaldata as provided in FIG. 1 indicates that secreted proteins of thepresent invention are expressed in the testis, nervous tissue, fetallung, brain anaplastic oligodendroglioma, lung carcinoid tissue, softtissue leiomyosarcoma, ovary tumor tissue, and germ cell tumor tissue.Specifically, a virtual northern blot shows expression in the testis,nervous tissue, fetal lung, brain anaplastic oligodendroglioma, lungcarcinoid tissue, soft tissue leiomyosarcoma, ovary tumor tissue, andgerm cell tumor tissue. For example, the kit can comprise reagents suchas a labeled or labelable nucleic acid or agent capable of detectingsecreted protein nucleic acid in a biological sample; means fordetermining the amount of secreted protein nucleic acid in the sample;and means for comparing the amount of secreted protein nucleic acid inthe sample with a standard. The compound or agent can be packaged in asuitable container. The kit can further comprise instructions for usingthe kit to detect secreted protein mRNA or DNA.

[0151] Nucleic Acid Arrays

[0152] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIGS. 1 and 3 (SEQ IDNOS:1 and 3).

[0153] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0154] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0155] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0156] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.) which is incorporated herein in its entiretyby reference. In another aspect, a “gridded” array analogous to a dot(or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0157] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and cDNA is produced andused as a template to make antisense RNA (aRNA). The aRNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0158] Using such arrays, the present invention provides methods toidentify the expression of the secreted proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of thesecreted protein gene of the present invention. FIG. 3 providesinformation on SNPs that have been found in the gene encoding thetransporter protein of the present invention. SNPs were identified at 40different nucleotide positions, including a non-synonymous coding SNP atposition 124. Changes in the amino acid sequence caused by these SNPs isindicated in FIG. 3 and can readily be determined using the universalgenetic code and the protein sequence provided in FIG. 2 as a reference.Some of these SNPs may affect gene transcription. Positioning of eachSNP in an exon, intron, or outside the ORF can readily be determinedusing the DNA position given for each SNP and the start/stop, exon, andintron genomic coordinates given in FIG. 3.

[0159] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1(1 982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0160] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0161] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0162] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0163] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica Such containers allows one toefficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified secreted protein gene of the present inventioncan be routinely identified using the sequence information disclosedherein can be readily incorporated into one of the established kitformats which are well known in the art, particularly expression arrays.

[0164] Vectors/Host Cells

[0165] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0166] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0167] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in prokaryotic or eukaryoticcells or in both (shuttle vectors).

[0168] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0169] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.coli, o the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0170] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0171] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0172] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0173] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0174] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0175] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptonyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0176] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enterokinase. Typical fusion expressionvectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. Examplesof suitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

[0177] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990)119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0178] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec1 (13aldari, et al., EMBO J.6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88(Schultz et al, Gene 54:113-123 (1987)), and pYES2 (invitrogenCorporation, San Diego, Calif.).

[0179] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165(1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

[0180] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J. 6:187-195 (1987)).

[0181] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0182] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0183] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0184] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual 2nd, ed, Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0185] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0186] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0187] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0188] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0189] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such askinases, appropriate secretion signals are incorporated into the vector.The signal sequence can be endogenous to the peptides or heterologous tothese peptides.

[0190] Where the peptide is not secreted into the medium, which istypically the case with kinases, the protein can be isolated from thehost cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0191] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

[0192] Uses of Vectors and Host Cells

[0193] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga secreted protein or peptide that can be further purified to producedesired amounts of secreted protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

[0194] Host cells are also useful for conducting cell-based assaysinvolving the secreted protein or secreted protein fragments, such asthose described above as well as other formats known in the art. Thus, arecombinant host cell expressing a native secreted protein is useful forassaying compounds that stimulate or inhibit secreted protein function.

[0195] Host cells are also useful for identifying secreted proteinmutants in which these functions are affected. If the mutants naturallyoccur and give rise to a pathology, host cells containing the mutationsare useful to assay compounds that have a desired effect on the mutantsecreted protein (for example, stimulating or inhibiting function) whichmay not be indicated by their effect on the native secreted protein.

[0196] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a secreted proteinand identifying and evaluating modulators of secreted protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

[0197] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the secreted proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0198] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the secreted protein to particularcells.

[0199] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0200] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/toxP recombinase system of bacteriophage P1. For a description ofthe cre/toxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0201] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0202] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, secreted protein activation, and signal transduction, may notbe evident from in vitro cell-free or cell-based assays. Accordingly, itis useful to provide non-human transgenic animals to assay in vivosecreted protein function, including substrate interaction, the effectof specific mutant secreted proteins on secreted protein function andsubstrate interaction, and the effect of chimeric secreted proteins. Itis also possible to assess the effect of null mutations, that is,mutations that substantially or completely eliminate one or moresecreted protein functions.

[0203] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 4 1 848 DNA Homo sapiens 1 tgggacaaca caggtcgcgg aggagcgttg ccattcaagtgactgcagca gcagcggcag 60 cgcctcggtt cctgagccca ccgcaggctg aaggcattgcgcgtagtcca tgcccgtaga 120 ggaagtgtgc agatgggatt aacgtccaca tggagatatggaagaggacc ggggattggt 180 accgtaacca tggtcagctg gggtcgtttc atctgcctggtcgtggtcac catggcaacc 240 ttgtccctgg cccggccctc cttcagttta gttgaggataccacattaga gccagaagag 300 ccaccaacca aataccaaat ctctcaacca gaagtgtacgtggctgcgcc aggggagtcg 360 ctagaggtgc gctgcctgtt gaaagatgcc gccgtgatcagttggactaa ggatggggtg 420 cacttggggc ccaacaatag gacagtgctt attggggagtacttgcagat aaagggcgcc 480 acgcctagag actccggcct ctatgcttgt actgccagtaggactgtaga cagtgaaact 540 tggtacttca tggtgaatgt cacagatgcc atctcatccggagatgatga ggatgacacc 600 gatggtgcgg aagattttgt cagtgagaac agtaacaacaagagtaagta actgcccggc 660 tccgatggtc cccgagagag gagcatggag ggaagttctgcctgtcacct gtcttcttgt 720 cgactcttct gcgccatgct gtgtcccgcg gcccttgcctttccccgctg tgtctacttt 780 cctgactttc aaacctgaga ataaaccagt gttgctgcacagccaaaaaa aaaaaaaaaa 840 aaaaaaaa 848 2 172 PRT Homo sapiens 2 Met GlyLeu Thr Ser Thr Trp Arg Tyr Gly Arg Gly Pro Gly Ile Gly 1 5 10 15 ThrVal Thr Met Val Ser Trp Gly Arg Phe Ile Cys Leu Val Val Val 20 25 30 ThrMet Ala Thr Leu Ser Leu Ala Arg Pro Ser Phe Ser Leu Val Glu 35 40 45 AspThr Thr Leu Glu Pro Glu Glu Pro Pro Thr Lys Tyr Gln Ile Ser 50 55 60 GlnPro Glu Val Tyr Val Ala Ala Pro Gly Glu Ser Leu Glu Val Arg 65 70 75 80Cys Leu Leu Lys Asp Ala Ala Val Ile Ser Trp Thr Lys Asp Gly Val 85 90 95His Leu Gly Pro Asn Asn Arg Thr Val Leu Ile Gly Glu Tyr Leu Gln 100 105110 Ile Lys Gly Ala Thr Pro Arg Asp Ser Gly Leu Tyr Ala Cys Thr Ala 115120 125 Ser Arg Thr Val Asp Ser Glu Thr Trp Tyr Phe Met Val Asn Val Thr130 135 140 Asp Ala Ile Ser Ser Gly Asp Asp Glu Asp Asp Thr Asp Gly AlaGlu 145 150 155 160 Asp Phe Val Ser Glu Asn Ser Asn Asn Lys Ser Lys 165170 3 33239 DNA Homo sapiens misc_feature (1)...(33239) n = A,T,C or G 3actgtgcaga cctgtcgcct taggtctccg ccttccagag ttttggggag ggggctgccg 60tggggtttgg acctggaaat ggctgaaatg caaatttcta gtgccagcta gcgggatgat 120ggaacggctt gagattcagc ggagcgccct gtccaccttt ctctgcctgg agccaaggag 180gctcctctgg gcagggacag gtgggcgagc aggccgggga aggagcctgg ggtgaaacct 240ctgcgaggac ttaggattgt tcctgaagtc gtgttagagg tagaaaattc ccctgcattc 300tgcaaatgct tagtaaatca atgagaatgt ttgtgtactt tcctcccaca tgtatgtaga 360aatagatccg aaattatctt ctgaggctct tgactaagag gaaagaaaag ttaagaggaa 420aaatctttca agctatctga aagcacatgc acacaccgtg cacgcacaca ccatgtacac 480acacacaccg tgcatacaca cacaccatgc acgcacacac accgtgtaca cacacacaca 540ctgcacacat acttttcttg cttttgctgt gaatcacacc gtttcacctg tgctatcttt 600gaagcagagg ttttctttat tcctcctttg gaaatgatgt ggcatattct tttccctcct 660ccatttttgt cctcagtcaa agctaaaagg agcagttttt atgatgagat ttggggaggc 720tccagcagtg ttgaaatcct atgacataac ttgaaacctc gagtgggtac ataaaaaatg 780tagagtttag agatttttgt attaaaggcc tccctgccac ccccagtctt agaaaatgtg 840gttcattcct gtggttcaca gaatctgaag cctgagattg atgagcccct tcttgtgatt 900gttttaatca ttctctaaag tttgtgcatt ttactgtacc cttagtcaca gagagtactg 960agtgaattta aagttgcttg gaatatatta agctttcttg gaaaattctc tttcccttgg 1020atcaaatggg tgaacagaat attaagttga gtgtcctctt ctaacttata ctctgaaaat 1080ttagccagta ggtttgcata tggaaagtta ttggtccaat cacataccat gactataatt 1140tacataattt acatcatcat tattaataaa ttcctgcatt attcatcaga atttgcatct 1200gctcagtttc ctcacccttc tacagaggtt gaagacattt agcagccctg gactgccatt 1260tagtcacagg ggaagggaat tcaggcttcg aagtcctttt tggatcagct gcgttgaatg 1320cgctgcagtt gtgttcactc ttcgtaagtg gctgcttggc ggaagactga aagacacttc 1380tccatttgaa aatgaatcct gacaagtgta aactggtggg aattttctgt agccttcctg 1440ggattctttt gattttgctg gtctcctttc ttcccaagag cgacagtgag ggtgggagac 1500tgctcacctc cagcccagca gaaatacgga gctgtgagca gactcctcca acagtttagc 1560tttctaaagc ccagttggac taagagaagg gaacgaggtt ggttatgcca acccaagcat 1620ttttaacttg cttttgtagt tgcccttgaa actaaggtca cgttgcttct gctcattgag 1680ggcggtggag gcgatagtgg atagtagaag agagcacaca ttgcctcact gaagtggctg 1740cacgtatctg agtcctgtag ctactgtttt atctctgttt cttaaaagta tgcttttaaa 1800aagattagcc tcacacattt ctgtggaccg gtctggtggt atcacctggg actctgaggt 1860gaggatggaa ggatttagca gataatgaaa aagaactctg tttgcgcaca tttgagaggc 1920tgaaaaatgg ttttatccca cttgggctgg agtgatttgg cattggggaa gattccctga 1980ctcgccaatc tctttccttt agtgactgca gcagcagcgg cagcgcctcg gttcctgagc 2040ccaccgcagg ctgaaggcat tgcgcgtagt ccatgcccgt agaggaagtg tgcagatggg 2100attaacgtcc acatggagat atggaagagg accggggatt ggtaccgtaa ccatggtcag 2160ctggggtcgt ttcatctgcc tggtcgtggt caccatggca accttgtccc tggcccggcc 2220ctccttcagt ttagttgagg ataccacatt agagccagaa ggtaagtcat ttaatttcac 2280ttttcaggtt tgttttggga tttgtctggg ggcagattgt taaggcctgt tttagaatca 2340gctacccttg cattgtaaat ggggcttcta agagcaccag atcgtggtct cttggctccc 2400ggcaaggcag agctgatgag agaaggtcct ttgccgcagc actgcaggca ggatggtata 2460gtttggtggt ttcttgctgt gtgtgtttct ctgtgctggg tgagggagac agctgggagt 2520tggcctttat ccagtgcccg agagagctgt ggaagggatg aaccttggga ggatgaatgt 2580catctctagt ctcccccagc atcattccta ctgcttacaa tgtaaagaat tgctattttc 2640ggacaagatg aaccactaaa agaaattgcc aacatctgaa atgctgacat cttattttca 2700ttgtttaagg agaaaagaag tttctgaagt ttagcacaga gagagaggct tgacatctca 2760gaattttttt ttagccattg taattcataa tacttaagca atgcatcatc acagagtggc 2820agattctagt ttaggtaaac ttgaaagtga atgtgccagg ggatctcctc cagctccgag 2880aacctccaag tatcacaggg catgcgccgg tgtcacagct ttggaaattt aattttcaag 2940gtgtaagtcc aattacgagg actacatgag gctgaattat tcagcttagc agatttggaa 3000cctctctccc agccctttgg agacaacgtg agccaagcct ctacttggtg ctgcactgaa 3060atctgtcatc agtagggaat attggtagct gagttatttt tcgagtggta atccgagaat 3120aaaacggcag atcccagcac tcatcgccac ttaatgaacc tgtttgcgga gagtccacct 3180ggtgcctgcc tggctttagg aacccgcagc agtccgagtg gtgtctgggg taagctgagc 3240tgctctggga acacatctcg tgcgtggggt gaatgaacag cacacttacc cagtggggta 3300ggctgggaga ggacagagag cccagcctcc ttagctggat caggacagtt taggaaggag 3360ggttgcgtcc atctgagatg agagttctga gagacatggg ctccccagga agaccccagg 3420cacttgtcat tgaaggatga ggaccgaagc acttatcacc tgaagcaatc gtgtgagact 3480ggggaacttt tcgttacaca gagggatggc agctctataa ttaacaggtg tggtgacatc 3540tccctacgtc tctgggaagg gaatctggta ggagtttgtg tctgaggctt gtatagctac 3600agctacaggc cgattatgga actgcctctt ggtgatttgt gctgggacac caggatgagt 3660gtaatttgca gtggtgatgc atttttacag ggctttcatg taagagggaa aaagttcctg 3720gtttctgaaa gtcatgcctg tacgtcttta attttgctaa taattaaaat ggatgtttcc 3780gtgattgctg gttttccctt gagtgcgtgg ctcggcaaaa actgaggaag ctgagctggg 3840atttcctacg gtgtgggctt taggagcaac ccaggttagg gaaaaactgt catttttcat 3900tttggctttt cagggcagcg ttattgtgag tttatttcaa cataaagtgt aaaatggtcc 3960cagaggattt gttttactgt atttaattct tgaaggaagt ttaaaattgt gtatacatgc 4020agggagggac aggtgaggca gagggaagaa atcgtggaaa gggacgaagt tccccgagtg 4080agtctttgtg taagaattta catccaacaa tgtgggtatt gttggaagca ggcctgctca 4140acctgggatc tgtggatgcg cttatggatc aggaaggagg ttgatgaagt tctggaaatt 4200ttaggtaata ttttgttaat gtccttttgt gcatttttat gggtttatag tttccatgag 4260ctatgagttc atttgcatcc agaattatga gatggaaaag aaaagatgtc ttagaggaag 4320gtgcaaaacc tggcaaagaa gaggctttgg aaaagtatac aagtcccagg cctcagtttc 4380cctttctgtc gtctgagggc ttaagaagat gatctttagg gtctcttcat taatagtcat 4440tgaatattaa tgtcctcaga agtcttctga ctcttccacc aaaaggttgt taggagattt 4500ccatttgacc tggacataaa gagcaattag cacaggggct ggcatccagt aagcaccggg 4560tgcatggcct tgtcgccgtt gtcgccaggg agctgggaac atgggtcttc ccagtcccgt 4620gggctggctg tgccaggtgc cacaatgtca aaaaacatct aggcttttgg agacagttga 4680gaagaaagtt gtttttggat ggaggaggcc cttggtgttt ccaggaagct gtgtttgctt 4740tctgtagggt ccccacttcc cctcatctgt tagtctaata ctggccactg atttgagccc 4800taagaccagt tcttctgttg caggagttcg ctttggtgtc agaggtgatt aggaaggtca 4860ttgaattata gatgagaaag gagtttttaa cagctgaaaa tgggctcagg tttaggctct 4920gtgctggtga attggaagag agagagagag agagagagtg tgcgtgtgtg tgtgtgtgtg 4980cgcgagtgcg cgtgcgtgcg tgcgtgcctg tgcccctgtc catcagttct ccatgattag 5040aactactata gctttggtta gcagtaagtt ccaccttgac cttctgtgca ccaaggtctt 5100atcttgtaag actttttggt ttgctaattt atttggaagc tcagtttaga taatgtctat 5160tggataggag aaaaatgtga ctgagaagtt ccaggaagaa gcctgggccc taacactaga 5220gggtcctttt cttttggccc ctcagggaaa ggttatgttt agtcatcgtg gctttgtgca 5280atatcgtatc atatcatatg tcatatcata tatcatatat atcatttgtc ttctggacac 5340gttctgaaat agtagcagtg gggttgggcc cactcaattg acttaagaaa ataatctcta 5400gaagatttta gtttttaata tttcagatta aatatgagtt ttccagttgg tcattcattg 5460ttaatttcct tttggtcatt cattgttata gggcaccagc agaggagtga aaacatggtt 5520attgactcga aggggggcag tcctcaatgc caagtactat tctgttgatt aacattataa 5580atatttgaag tctagtcaag cttgattacc ctgagagggg gcaactctta ctttcaagtt 5640cttcctgaca ttgtcaccta ctctggttaa ttaagtcatt gttgacatta atggcatctt 5700tgtttacacc aacccagtag gagctaaaat gaaagggctt ttcaacccta cacccttaat 5760tactttccca ccctccacag agtgtgactc tgaaaagtaa caacctgaaa aaaaatgtag 5820tttgtgtagc aattttttgt caatcttctg agaggtgatg ctctcctgga ccaacgtgag 5880ttggtccaga gttcagtagc agaaacgtcg aggacaggat ctacacagag acctccctaa 5940gtcagatttt tccagtatgg tttaagtcct ttgttagaag tattgtaaat gccataatat 6000taaccatacg catttaattg caagttaaaa gaaaggaaga aagaatatat ccgcatacca 6060agagtgaatg atttaaaata atcttctgtt tcgtattatc tgcatctttg tttttcaata 6120tgagtgttaa tatttaagag ttgactgtaa cttgatagtt agctttggaa caaggactta 6180ttcttggtca attaaaccaa atacaggctt acgcagttaa atacacaatg aagtacacat 6240tctttattag tatataaagt gtttcacaat tcatagacca agagccatgt ttaatattac 6300ttatagagca gaaatctggc aagcccagag aactggtact tgtatcattt tatactggct 6360ctctctgatt caaatttggg gtatatgtgt gtgtttctgt tttgtctgtt tatttcaacc 6420agttaaaaga cagagcactt actatgccaa tggccccgca tcaagccata agtaaagact 6480ttattccttt caagtctttc tgacccagtt gagggtgaga attcacaaaa ccacagcaat 6540ccaatatgag atgtttttaa tatcagactt aacaaataat tgcatggcta tgaaataact 6600ggggtcgtgt ttaaactgga agtgttttgt ttaatgttcg tagtttcaat aaaatgtatc 6660cactagtctt ccagtttgca gactgttgtt taggtgtttg tttagccagg gtaattgtta 6720aaaactccct ctaatctagc ttacccttac atttccatgg aagcgaattt tagtcattaa 6780aggaaaacat gggaaattga tttttgggtg cctggctgtt acgctaggta ggaaatatag 6840ctggtgtgct actctccact gtactggtcc gattctcgcc agggggacat ctctgtaggc 6900agttcagaaa ttattttttg gaagtttttt aggctattcc acagataatt ctgatccagt 6960aggttttatg caactgtgca aaatgcttat ggtctctatt tttttttcct tgagagatga 7020ttttagcctt tggttttttg tttgtttttg ttttttgaga cagagtcttg atctgccacc 7080caggctggag tacaatggtg caattgtacc ctcactgcat tgtcaaacgt ggctcactgt 7140accctcggac tcctgggctg aaggggtcct ccaacctcct gagtagctgg gactacaggc 7200ctggataatt tttaaaaata tttcgtagat atggggtttc gccatgttgc tcaggctggt 7260ctccaactcc tggcagcctt ggcctcccaa agtgctggga ttacaagtgt gagccaccac 7320acctgtccta gccttaagtt ttgcattttt ttccatcttt ttgctgtatc ccatatgatt 7380tagagatttt tgctgtatcc catgagattt agagatggct ctactttttt tagctttcct 7440agcattgaaa tgcttggtgt tgctaatcat accccatctt taccacacca tcttcctccc 7500tgacttgcct tcttagtgta gtttggtcaa gaacttgagg ccaagttctt ttttttttaa 7560atttagcttt tattttaggt ttgggggtac atgtgaaggt ttgttatgta ggtaaactcg 7620tgtcatgggg gttcattgta cagattgttt catcacccag gtattaagcc cagtacccaa 7680tagctatttt ttctgctcct ctctctcctc ccacccaccc tccaccctca agtagacctc 7740agtgtctgtt gttcttttct ttgtgttcat gagttttcat catttagctc ccacttataa 7800atgagaatat gcagtatttg gttttccgta tttggtttat tgctaaggat aatggcctct 7860agctccatcc atgttcttgc aaaagacatg atcttgctct tttttatggc tgcatagtat 7920tctatgttgt atatgtacta cattttcttt atccaatctg ttactgatga gcatttaggt 7980agattccatg tctttgctat tgtgaatagt gctgcaatga acatttgtgt gcatgtgtct 8040ttatggtaga atgatttata ttcctctggg tatataccca gtaatgggat tgctgggtcg 8100aatggtagtt ctgctcttac ctctttgagt aatcgccaca cggctttcca caattattgg 8160gctaatttgc actcctacta acagtatata aagtgtttcc ttttctctgc aatctcacca 8220gcatctatta ttttttgacg ttttattaat agccattctg acttgtgtga gatggtatct 8280catcgtggtt ttgatttgca tttctctagt gatgagcttt ttttttcata tgctggtggg 8340acgtatatat gtcttctttt gaaaagtgtc tatgtccttt gtctactttt tatgtggtta 8400tttgtttttc tcttgtaaat ttaagttcct tatagatgct ggacattaga tctttgttag 8460atgcatagtt tacaaatatt ttatcccatt acgtaggttg tctatttact ctgttgatag 8520tttcttttgc tgtgcagaag ctcttaagtt taattagatc ccatttgtca atttttgctt 8580ttgttgcaat tgcttttgat gtctttgtca tgaaatcttt gcccattcct atgtccagga 8640tggtatattg cctaagttgt cttccggggt tttatagttt tggatttgac atttaagtct 8700ttcttccatc ttgaggtgat tttttgtata tggtataagg aaggggtcag cttcaatctt 8760ctgcatgtgg ctagccagtt atcccagcac catttattga atagggagtc ttttccccat 8820tgcttgtttt catcagtttt gtcgaagatc agatggtcgt aggtgtgcgg taggtgtgtg 8880gccttatttc tgggctctct attctgttcc attggtctac gtgcctgttt ttataccagt 8940accatgctgt tttggttacc gtagccccac agtatagttt gaagtcaggt aatgtgatgc 9000ccccagcttt gttatttttg cttaggattg ccttggctat tcagaggcta agttctttta 9060aggagaggtc tgattgaaca agatgctgga caggtcattg tggtgatcct tcacgtcttg 9120aagatgtctc cttctgtaat agacaaaagt cacacttttt acaagtttct ctttcctcac 9180tgtgatttgt atgtggtagc tgacttctat ttatataact tcaagctctt accatttaaa 9240tatttataca caagtatgaa tcattgggac aagccatggc catccttgaa gagcgtgtgt 9300caagtaaaat aggcgtgctt tagattttca gtaattttgg ttttgggaaa ccggtaccat 9360ggaagagttt tcagatgctg aatgtgtaat ttactccact ttggaatact gtaagtaatt 9420ctgcgccatt gtggactttg gccacatcat gccactctca aaaacttctg attctttttg 9480acagtaccaa gtcagggggc taagctgttg tatattcttt cctttaaccc cttgtctaac 9540taaagaatgg taaattccaa ttcatttcag aatggaatgc acacatatag agtttcaggt 9600tatgctgaat gtgttttatg aggaggctaa aaatagctta taaggaaaat gctgatagat 9660tcaagaacga gaacaaggga tgtatttatt tgtatgattt atgtagcgcc tttcttggag 9720aggactcaat tctgagctct aacgttaata aactctatat caattatcgt gattattttc 9780gaacccttga gctcctatat tactatatgt gacccatttg tatacctgac agtttactgt 9840taaattttcc atagtgacca gttgtaatat tttaaaagta tccagacatt gaaaaggcca 9900actgtgtgta tctatggtgt gtgtatgtgc tcaggggggt gtactataaa atgaatgtca 9960agatcattgc aatggttaag tttaggtttt aaactctttg aacaccctta aagttcagat 10020tttcagttga cagtgggatt gcccccaaag atgtgtggct gccttaagga gccttcttgt 10080cctgctaagc ctcctggatt tccaccaatg catgttgcat ttctacctgg ctgcaataga 10140cgcacaactg aatatatatc gtggacattt ataaatgttt caaatttaaa accactcaaa 10200tttttaaaag aacaagaatc ttctgtgcat ttaatctaaa ttctgtatca gccattctta 10260aaaatagaat tgaataaaat cattttggat ggcatgtggt atgctttctg gagacataaa 10320ctaacagagg gaacttgacc ttgggaggta ggtttgaatc tctctactat ctactgatgc 10380tgtgatttca gaaaagttag ataacctctc tgagcctcag agatagtatc tatcttgaag 10440ggtgaatgtg cagattaaat gcaaatgaaa tgtaaagccc ctactgaatt tcctggccca 10500gagtaggtgt tcattaaatg ctgatttctt ccctgtccca cttcctgtca acatttcctg 10560gaccaacaag atgtttactc tacaatatta ccaatatttc ctggaccaat aagatgttta 10620ctctacagtg tatagtgata attgtctgat gagaagcatt tacattaatt aaaatgtaaa 10680actggctcta ggccctggat ttgtccaacc ctttttatta tcttgggctt ttgcaagtcc 10740catggatttt agaaatggga tcatcctgct gcttgcagtc caaatggttc aaggttgaaa 10800ttttttttcc cctctgttga aaaaagtcag ttgcagctct gtaataataa cagcagacac 10860atacttcatg aggaacaatg tattttggca aaagagtttt ctgtttgaag cttcaaaata 10920caaaatactc tgccacattg ccattaggcc cggtgaagaa cattggagag gggttatgga 10980atcggttggg gtggggtgcg ggatggagag gggttatggg atcggttggg gtggtgcggg 11040atggagaggg ttatgggatc gtttggggtg gggtgcggga tgtagagggg ttatgggatc 11100ggttggggtg gggtgcggga tggagagggg ttatgggatc ggttggggtg gggtgcggga 11160tggagagggg ttatgggatc ggttggggtg gggtgcggga tggagagggg ttatgggatt 11220ggttggggtg gggtgcggtg tggtgtgttc tgcttttggc caggctggag aggtgagctc 11280cataacatgg tatggtcatc cttgccatgt ttgcctgtag tgacctcccc tgaacactcc 11340tagatgagat ttttgtctgc atggaaggga gctcaaggaa attgtgatgg gacccacagt 11400actgaagtgg acaggagctg aacaagcatt tgcacatgat acccctatgg aggttacttg 11460gtaggacagg ctatgaagta ggggcagtat ggatgaggtc acgtttcttt agcccattcc 11520ttgagatgat gatgatgatg atggtgatga tgataactgg ctaccattta ttgagtgcct 11580gtcatggttg gactctgtgc taggtacttt aacatatatt atctctcatc ttaataaccc 11640agaacatctt gggttttcct gttcttattt tatggacaag gtaactgaaa ttcttgggga 11700tcaagtgact cctccaggac ctcatcatta gtgagtgtgg gcccaggtct ttgggtgaat 11760gtgttggtgt aagaaaaatt tcccttcttc ctgagcagga ggattttttt tttttgctgg 11820agaatgtggt gacccctcat tctttcctaa atctgtcgtt tgactattaa acctgttagg 11880gacactgctg gttgattctg tttctttctc acacatccat accgaattct cctaaagaca 11940tttgaagaaa aattccagac aataaaaatg tttaatgatt atactttgtg attcttctag 12000aatggcttct ggtggcatgt gacttattgg aaagaggcta accctgactg ctgccaagga 12060gaccaatggg agactgggtc ccagttggtg gtccaggctg cgccacaccg taggagtcca 12120taacaagaag ggctggcctc tgttgcccgt tgatgcatga gccatctcag cagagcaggc 12180cgtccccagt tattccttgt cccagatgcc tctgctggtt tgtcctagtg cctgctaacc 12240ctcagctgtt gccacttgga tattatgtca agcttttctt cccagaattt ctgatttacg 12300actgggtatg aagtcaaggc ctacctgcat agacagaccc ctgtacttgg gactcccacg 12360aactgtggtc atggaaacaa gcaccaagga ctacctgacc cttctaatgt cacttttctg 12420cagagattag gaggagagac taagaaagcc aaaaaagaaa aaattccaag aatgaaaagg 12480ccaaaagcag aaacacctac tcttcttcca gtcttggcca gaaagggtgt ccttagggga 12540aaaaaagagg ccagggaatg gagcccattt ttaaagcaag caaagatcag tttggtattt 12600aaaaataaaa agtgaactcc agtaccagct aatgtgtagg tgaatcgagg ctggtttagg 12660cagctgaact ttgttcttgg ctacctgtac agcagttgca gaacactcag gggttccagg 12720catttccaag tgggagttga gttttggagg aaagttgtag aattcctttt tctttttttt 12780ctttttttgt gagacagagt ctcttgctct gtcacccagg ctggagtgca gtggtgcgat 12840cttggatcac tgcaacctct gcctcgcggg ttcaagcgat tctcctgcct cagcgtctag 12900agtagctggg attacagatg tgcaccacca cgcccggcta attgtatttt tagtagagct 12960ggggtttcac catgttggtc tcgaactctt gacctcgggt gatccaccca cctcagcctc 13020ccaaagtgct gggattacag gtgtgaacca ccgcacctgg ccgtagagtt cttgactagg 13080agatgctgca aatttccctc ttaaatttgc agctccccgt gcgatgacaa ctttcagagc 13140ttggcagagg caagatggta taaacatgat ttttagattg cagggtaatt gctgtggttt 13200ctttgagttt tttcatccat ttgctcccca aataattttt gagacctact atgtgccagg 13260ttctgtgggg atggaatagc aagcaatgaa cagttgagtg ggggagacag gctcttgtca 13320aataattgca aaaatgaagg gctaggactg ggtgcagtgg ctcacgcctg taatcccagc 13380actttgaggg gccgaggtgg gcggatcatg aggtgaggag tttgagacta gcctggccaa 13440catggtgaaa cgtggtctct actaaaaata caaaaattag ccgggggtgg tggcaggcac 13500ctgtagtccc aggtacttgg ggggcggagg caggagaatc tttgaacctg ggaggcagag 13560gtttcagtaa gctgagatca cgccattgca ttccagcctg ggcgacaggg tgagactctg 13620tctcaaaaat aaaaaaaaaa aaaaatcaaa ataaataaac aaaaataaaa aatgaagggc 13680taggtccagt caaaggtgaa gattctgtga aggaaaagca tgggccggag gcatgtgccc 13740catgtttagg agttcccagg gactgggact atgaagctgc tgctgctgct gctttttttt 13800tttttttttt ttttttaaat gcagggtctc tctctgttca ctgtgtcacc caggctggag 13860tgcagtggtg caatcacagc tcattgcatc ctggacctcc tgggctcaag cggtcttccc 13920accttagcct tctgagtagc tgcgactata ggcgcaggct gatttttctt ttttctttct 13980ttttttcttt tttttttttt ttggtagaga tgggttctca cttgttgccc agcctggtct 14040caaactcctg ggctcaagcg atcctcccag agtgctggga tcacaggtgt tagccaccac 14100acctgaggct ggtcttcccc ttaaggaggt ggcacccacc tggattcccc agcaaaccca 14160cctgcattgc aaggttgacc cttatcagta ccagccacct tgcctgctag gttgaccctt 14220gtccagtgag gtgattttcc agggcctagc ctctctgctg tcccttgctg gcttcacctg 14280ttgatgttga tggaggtgga gcagaggccg ttgagtgaat gcgtgcagct gggctcagag 14340gcccctcttc tcccctcctg tgaggtgctt gcccttgaag gtgtggcgag tgaggaggcc 14400ggtcaagggc atcccggcgg cctccaggcc gtatttgagt gggtcgtttc agcctgcttc 14460ctatctcttt tctgttacta cctctaattg gcagagtttc ttgccaggtc aatgtggagg 14520cagagagatg gccggagggc ggccagggga gtcaggccag gtgtgggcag gatgggattc 14580tgcctcctcc caggtgcctc gcctggggga tgccctgtcc cagaaagcct acattcgtgg 14640gagccggcgc acagcccttc tgagatctaa agcttccctc tgaatgctgc tttggaggat 14700tgtgagaggt agtgactctt caaagtttgt ttgttttctt gaagctttta cctctatgca 14760aatatgcagt ttggagcagg gaagaaaggt taactgtgat ggcgccggct cttaacgtgg 14820aatgtcctga attaatgtgg gtttcagtcc tctggctcag gatcccctga gggagagttt 14880ttctttcctc tgcaaaacac aggagaaaag tgatccctgt ggctccgacc tgccttcctt 14940gggtcctgcg gtgcaaaacc agctgggacc gtgtcccgcc cacccgaagg cagtgtgggg 15000aacctttcct ccaggtcatt cccattcagc tgattgctgc cggctcccca ggccacaact 15060ctgtgccttc aggcgtctgc acgggtttcg atgctggcca ggcctgaact tggtgagcct 15120caagcagacc gttcaaaccc attcaaatga ggaagaccat ctgtttccca gtctccagct 15180gctgctgctt catttgcaaa tggctgggat gctgctgagg ggatcaggcg gggacacatc 15240tgcagactct gaaggagtgt tggaaccgag atcctgctga gagaagaaag gccgagccct 15300ttaaatcaac ttgccaaaca gtacccccag aaggtcctga gttgagaaag caggaggcag 15360ccttgccctc ctggaataac tcttaacctt cccttttctt ttgtagcctt ggccacttta 15420aaagtatttc tttattcaga aagtgcgcag tgtgggaggg cctgctctat gggcttgggg 15480gaaaatgtca aacgggatct ggacatctat ctgacctttc agggccatac agggcaaacg 15540tatccgctgg agtatgcacc atttattgaa tgtttacatc aatatcaggg aggtgagctt 15600gtcccagcag cagcttctag gagccacagg taacagtaag tgtggcaagg tgactgtccc 15660tgaaaacctg cttctggaat gagtcaggct ttagggtatg ctctctggaa tgcaggccag 15720ccgccccaac tcgcagtaac gcaggccctt agctctgtgg actgcgtgag gcacagctgt 15780ggggactctt gcccatggtt tggtgtttgc agggttattc ccggcatgct gtggggctag 15840ggtaagttat ccggctcctg agccctgctg gggttctcat ctcaagggaa ttctgtggtg 15900tgttactgtg ccccacatgc aaatatcagc tactctcaaa tgtgttggat ggatgaatag 15960tagaaggtat tttaagaagc cacaggcctc tttgtaaatt aaacaggcat catacatggg 16020tgttgataat gatgaatctc acaaaatctt cagatgttta gtctctggga acattccagg 16080aatcctcatt taggtaactt atatgtgatg agacctattt gttcacttga aagaaaacct 16140gttttgaagt cagaggaatg cgaatagagg ctctcacatg gttggaaaaa gcaatctgca 16200ggccagttac gccccgtaaa caggaaccca ggactgccct cctggccagg gctgagttgc 16260aggatgggga ccccccacta cctccaaccg cccgccagga tgaggagtgc ttgctctcag 16320acgtgcccct cactttaaat atacagaggc cttcctaggc agcctttgat tgtgtacctt 16380gtggtgacct tgccctgcag caggcagcac tggagatgtt tttttcttct ttaaagcatg 16440actctgaggc tcagcggtgt gaggctgtcc aagctgacac gttcactact ggcagaggcg 16500ggtctcaaag tctcatcctt ggacactgga gctgaacttc ttgtagtgtg ggtcttggac 16560cagcagcatc aggcctcacc tgtgggaaat aaagaatgtc agcccgcacc tgcaggccta 16620ctgacccaga atcttttttt gttttttcct tttgagacag acttttgctc ttgttgtcca 16680ggctggagtg caatggcaca atcttggctc actgcaacct ctgcctccga agttcaagtg 16740attctcttgc ctcagcctcc cgagtagctg ggattatagg ctcctgccac cagacctggc 16800taatttttgc atttttagta gagacagggt ttcactgtgt tggccaggct gttctctacc 16860tcctggcctc aagtgaccca cctgtcttgg cctcccaaag tgctgggatt acaggcgtaa 16920gccacagcgc ccagctgacc cagcatcttt ggaagtggga gcaaggaagc tgtcttaaac 16980tgaccacgtg gttttacgca cagtaaagtc tgagaaacat tgcattgatc cctactccag 17040tccctctccg tacacctttt gggtggagtg ggctggggac gcagactgtc tttggctgtg 17100catgtcctag aggctgaaca ggacgagatg ggagcagtgc agtgtcttaa tgggaatcgg 17160gattttcacg gaggagctgt tggaactggg ctgaatagga gcctgccagg cagcagagct 17220gggtgggctt gtaggtagag ggaacagtgc ccatgtggac aggtaagcca gagtgtcaga 17280gagagggatg tggggcttgg agcaaccaag cctcaatgcg catgatgttc tttgggtttt 17340attctctctg actttggagt ggtctgtgcc tttttaaaag agtagagatc atgtgctttt 17400cagaagattc ctctgggggt ctgcggtggc tacaagaggc gcaccatggg tgatgggtta 17460ggcgcattgc agaggtcttg tggaggactg gaggagacct gtgggatgca gttttgcctg 17520tactttcttt cagagctaag ctttctatca gggataggcc taataggtga aggggtgtgg 17580ggactgatca gaggaagggc cagaggaaaa gaggggcttc agggccgact tggagcttgg 17640gcggcagttc agtggtgtga ctcccttcat cgtgtaagag aagaggctgg tggaggagga 17700ggaggttgaa ggtcacttgc ttgttttgga tacgacctct gcagacatcc aggttatgta 17760tttcctcccc ccgggcaggt ggaaatatga acctacaagc agggacttga gtggcatctg 17820cggggaggag gtgggaaaag ccacacgtgc ccaggagact ggaatgcagg gaaaggacca 17880gaagagccag aggtggaatt ctgggtatat ccatggatac aggaggggtg gcagggaagg 17940agaaatttcc tagaaaggca agaagtcctc cttgacatgt tcctgtccat aagaacacat 18000acgcacatgt acgcaccagc aggaagcaga atgctaaccg aagataatta acccccaatt 18060ctgtgttagg gattgagaaa tagaccagga gccctgcccc ctcctctctc atttcctgac 18120cttccacact gagaagacct ggctaggcag ccttgctttt tttcctgttt agcggaggag 18180tgaggatttc agccggaagg tctttctgat ggcagatgtg taagtgccag acattgtgct 18240gggtgccttc tgtgtcctat ctcatttatt attgttcctg ctccgaggac ttgcctcaag 18300gtcatacgat ttgtaagtgg catagtctcg gtgtcagtga caggtctgtt tggtgtctct 18360ctctctctct ctctctctct ctctatatat atatatatat atatattttt tttttttttt 18420ttttttagag atggagtctc gctctgtcat ccaggctgga gcacagtggc gtgatcttgg 18480ctcactgcaa cttccgcctc ccaggttcaa gcgattctcc tgcctcagcc tcccgagtag 18540ctgggactat aggcgcctgc caccatgccc agctaacttt tatattttta atagagatgg 18600ggtctcccca tgttggccag gctggtctcg aactcctgac cttgaatgat ccacctgcct 18660cagcttccca aagttctggg attacaggca tgagccacca tgcccggcct tggtctgtac 18720ctttaacacc cccagccttt tctgaagagt caccagagaa gggacaaaaa tgaggccata 18780gccttactgc taagggacca tgagaggctt ggggtatagc tgtctgttga gacaggtgct 18840ttactacttt gtaagatgaa gagagctgcc tctggctgag cactgtcatt aggactcagg 18900gaatggaagt gttttgagac cagagggttc agtttcagga ctggagatca caatgcattc 18960attttacaga gtgacaactc tgtagggcca ctcttcaccc taagattggg tcattaaagg 19020ccaggcacat cctattcact ccttcacctc cttgtgagcc ccccacatgc cttttgatga 19080aagggttttc cccagaacag tgtgtcccaa gaagccctga agggctggag atgtaccagc 19140tttctctgct atgtccagca agtgtatttt cagaaggtag gaggctcggg ctgggctggc 19200caggcagcca ggcacacaga ctcctcattg tacatccaag ccggggcgtg caggacttca 19260acatagcttg taacgcaagt atctatttcc tgggcgctac atgatctaat ggcctgtcgc 19320tttgggaaat gctttctgaa caaaagactc gatttattta tttatttatt ttttgagacg 19380gagtttcgct cttgttgccc aggttggagg gcagtggcgt gatctcggct cacggcaacc 19440tccaccgccc aggttcaagc gattctcctg cctcaccctc cttagtagct gggattacag 19500gcgtgtgcca ccacgcccgg ctaattttgt atttttagta gagacggggt ttctccatgt 19560tggtcaggct ggtcttgaac tctcctgacc tcaggtgatc caccctcctt ggcctcccaa 19620agtgctggga ttacagatgt gagccactgc gcccggcctt ttattttttt aaattattat 19680tatttttatt tatttattta tttttttgag atgaagtctc gctctgtcac ccggctggag 19740tgcagtggca cgatctcggc tcactgcaac ctctgcctcc ctggtttaag cgattctcct 19800gcctcagcct cccgagtagc tgggattaca ggcgtgcacc accatgcctg gcttaatttt 19860tgtattttta gtagagatgg ggtttcacca tgttggccag gctgctcttg aactcctgac 19920ctcaggtgat ccacccgcct cggcctgcca aagtgctggg attacaggca tgagccaccg 19980cactcggcca ggactcaatt ttgaagttct tatgcaagca aagctgccca tatctaggag 20040tttatgcaca cagtactgat tcaatacctg cgtcttagtg gtccatcagg aatttttcac 20100tcatgaaata tgactttaat acacgtgttg ggaccagaga gaaaaccttc cctctgtgca 20160tctttttttg gagaacactg tcatgaacag ccgaaaacat cagatcaaaa gcgagaggga 20220tactgacaaa acagggagtg cggatttcca ctgtgggtgg ggacattggg agacacggtg 20280actcactcct cagtaagtgc attcttaggc tctttactct gtgtgcattt ttattttctc 20340atgggcaaca atataatttc tttcaaacat gaagagccat ccagcatgtt tctcaaaggc 20400agacttaaat gatatgaggg gtgtgcctaa atatataatt tttaatggtt accatattgg 20460aaacattagc catgatccca tctgatgact aggaacatga gccagtctgg agtttctgga 20520gaaatccagc taccgctgca gaggcggctg ttagctgttg ttaccggcat ccttgttagc 20580gaccagggag gtttcagtcc cgacttgtgt ctaccagata ccctgacaac tggttagaaa 20640gagaggactg caccctttca tcctgcgtac ttatctgttg tttgttgctt tgacttcttt 20700ttgttctccg tttttatgtt ggcagtattt ctcgaggtag agaactttca cctttatatt 20760gtgcggagta tttgtccctt cctcgccccc ttaaagaaca cgtagtacct atgccttata 20820aggtctctgt ttgatgtgag gaatttggtc ttcttgcaag tcggctcttg cagaggggag 20880gctttgagga tgtctgggct gcagcaggct gtcttcgttg ctgtgtacac agccctcatg 20940gcagggcatg caaaggtggt tggttctgac ttcaatggcg ggactccatt gcctttattt 21000tttaatttaa ttattattat tattattatt tttgagatgg agtttagttc tgtcacccag 21060gctggagtgc agtggcgcaa tctgggctca ctgcaacccc cgcctcccgg attcaagcaa 21120ttctcctgcc tcagcttccc gtgtagctgg gactacaggc acacaccacc atgcccatct 21180aatttttgtg tttttagtag agacggggtt tcgtcttgtt ggccaggccg gtctcgaact 21240cctgacctca agtgatccac ctgcctgggc ctcccaaagt gctggtatta cagacatgag 21300ccaccgcacc tggcctccat tgcctttatt tctttctgct gataagttct gatgccagtg 21360atacccagat tgtgccatag gaaagagggg gctgggctct tccagaaacc ctatcttgca 21420gaaatctctc ttctgttcct acggacagag aattgggtat tgaccagtga ggacttctta 21480ctgggactgg gcgtggccac tacaggacat cttccagact aagagggcca gttgggggtt 21540actggaccag ggaggacagt ggcggccact tacttgccat gtgctttgtg tgcagtgact 21600atgtagcgac atttgcagcc ggactgtgtt tctcctgtag agacactggc agccctacag 21660ccctacagcc gtgacattta ctctgtacaa ggtggcagga ggtgtgggag ggcaccggga 21720accgaaggcc attttactac ccttcccgca gcgctcctgt tacagtgctt gtgggagtcc 21780cagctgtgcc tccaggtaca aacgggtttc ttcctgcaac ccacaaccct gacaggacat 21840gcctcccggg tgtgctccta acctgcctct tctctctgcc cctccctctt tctcctctcc 21900ccttcccctc tctttcttcc tcttgccttc ctcttctttc ctctccctcc ctctgtttct 21960ctcccttcct tttccttgtc tccgtctctc ctctcctttt tccccctctc cctctctttc 22020atccttctgt ctcttccaga ggagtctgtt caggggattt ctttctctct ttcttcttcc 22080tggcaggaat gtgtttaggg attggacacc tctgactttg gaggagggag aaacctcagt 22140gggatgggtt ccaggacgac cccaccgcat ccttggaact aggatgatct agacgttgga 22200aaagacacca tccctgggaa accccagaaa aggcttaatt tgtgaaaagt aatggaggga 22260gctgtgccgt tggtagaaac tgcttttttc ttccttaaca gtttaaatct gtcgtccatt 22320ctccgtgaag tgattggacg gggcaagact caggtttccc atctgttctc tgtttgcatt 22380tgggcgccat ttcaaaaacc acacgggaaa agtttatagg caaacattat aaaaagtgac 22440agtctgaagt gctgctatcg ctggtttggc aacgtaaagt gttacctgaa atagcttacc 22500atttccaaac ccttttgctg tttcaactgt ctcaagacaa ccctcccgct gagatgggtg 22560agaagtccag ctggatgtgt gcagtgaagt cacataagtc acagcctttt ttgactttta 22620caagatttcc ccctcctggg gctatcttca cacacagcga ggattttttt ctctctgttt 22680acttatagag aggtaaattc atgcagcttg tggctagtgg cactctgtgt gatgtcaaat 22740ggtctgctga ggggctcgga gagtcaggca gcccctgcct cagtttcttt ctctccccag 22800tcagaggtcc tatgagctcc caaggaataa caggatggtt ttcttaggga aggaaggcca 22860ggtcaaggca ggaattactc acagctcatg tgcagatgcc tgttgttatt catactattt 22920atttatttgt gttttttttt tgtttgagac agtttcactc atgtcgccca ggctggagtg 22980caatggcatg gtctcggctc actgcaacct ccaccttctg ggttaaacga ttctcctctg 23040cctcagcctc ccaaatagct gggattacag gcacatgcca ccacgcccag ctaatttttt 23100atatttaata gagatgggtt tcaccatgtt ggtgaggctg gtctcgaact tctgatctca 23160ggtgatccac tcgctttggt ctcccaaagt gctgaaatta caggcatgag ccactgtgcc 23220tgggctactt atttattttt ggagacaggt tctcgtctcg ctgtgtcacc caggcaggaa 23280tgcagtggcg cgatcatagc tcagtgcagc ctctggggct caagcgatct tcctacctca 23340gccccctgtg tagctgggac tacaggtgtt tatcatcatt cccggctttt tttttttttt 23400tttttcattt tttggggaga gaggatctta ctatgttccc taggctggtc ttgaactcct 23460gacctcaagt gattctccca cctcgacctc ccaaagtgct gnnnnnnnnn nnnnnnnnnn 23520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 23940nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24000nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnngaagac 24060accctgtctc tactaaaata caacaacaac aagagaattc ccaggcgtga tggcatgcac 24120ctgtaatccc attactccag aggctgaggc aggagaatca cttgaaccca ggaggtggag 24180cttgcagtga gccgagatcg caccactgca ctccagcttg ggcgagagcg agactccgtc 24240tcaaaaaaaa aaaaaaaaaa aaaaaaggcc aggcacagtg gctcacgcct gtaatcccag 24300cactttggga ggctgaggtg ggcgtatcac gaggtgagga gatcaagatc atcctggcca 24360acatggtgaa acccaccccg tctctactaa aaaaaatacc aaaaattagc caggtgtggt 24420ggcaggctcc tgtagtccca gctactcagg aggctgaggc aggtgaatag cgtgaacccg 24480ggaggtggag cttgcagtga gccaagatcg cgccactgca ctccagcctg ggcgacaaag 24540ccagactccg tctcaaaaaa aaaaagaaaa aaaaggctgc ttgctaacac gctgctggta 24600cttcccaagt atttgaaact tctgggagaa aattcacgtg gatgcaactg agctttatgc 24660agcagggctg cagtagtgtg gccacggcag tagaaacgag tgttgagctg tgtcccagga 24720aggagacttg gtgccttgtt tgtttaccag ttttaggaaa cagtgacaaa gattggccaa 24780tactactgtg aagcttgctt gcttcttttc gacttttaat tcatattgtg aaaaatacat 24840ggaaacatat tctctgtctg tggaagcctc tgaaagagca tttgtgcata gcaaggcggc 24900ttctggaacc ttctccccat tgcgttttgg caggagatga gggagatgtt gggctgtgca 24960cccctggtgg attcgcacag gaaaggtgaa aagtatgcca cagcaatggg aaaaagtaaa 25020tccaggaagt gctcacatga ctataaaagg agttatgtac atttcctggg gaattctttg 25080ctctttcaca gagcaggctg tgcagcctgg tggagtcagg gagaggttgt ccatccatgg 25140ggggtgacca gagttaggga ggcccctttg cacatgcagg cacaggggat tctctgattg 25200tcctggctcc gcgcagcctg gggccaggtc agtacaactg tggggatgaa catggagggc 25260tcagggagtg catgtttgca gggagtgggc agcgggtcag gtcacctgca gcattcaaac 25320ccagaagaga agctaagccc tgtggacggc aaatggaccc aaggacacgg gcagggacag 25380agctgcttcc actgagccag ctgttgtgct tgtgttcttg gccccctggc cggggttgtc 25440tttgatatgt gtcgtgtgtg ctgggccctg caggctgggc ttggggggcc actgggtggc 25500ctgggaggct accagtgtgg catttggccc ctccccagcg tatggcccca ccccatccct 25560gcctctcttc ttacttacat cctcctcttt ttattctccc caacctcttt ctctcttgtg 25620tgtttattca tgcattctcc ctaccatgtg cttgcagagt cttctgggat acccaggggt 25680attactggca tgtttactaa aggcaccgct gatccccaaa atgggccctt tgtgaaatga 25740ccactgaagg ggtggaggga ggagtccttg ctttctcagt ttaccaagac agcagttact 25800ggtttgagac tttgaagaca ggatggcttg atttttatgg ataaacatca ttatctcatt 25860ggtgtatttt agggggtgat attttactat ccttgaagct tggatatcta gatgacagtt 25920ttgatctggt tttactggga tcaaaagcag ttttagtacg tcaagtcatc ttaactgtcc 25980attgagatgg gactcctgta aaatcacttt gtatctggag atagcaatcc aagtgttctg 26040gctgggcgca gtggctcacg cctataatcc cagcactttg ggaggccgag gcgggcagat 26100cacgaggtca ggagttcaag accagcatgg ccaatatggt aaacgctgtc ttctcataaa 26160aatacaaaaa agttagctgg gtgtggtggt gcacgcctgt aatcccagct acttgggagg 26220ctgaggcagg agaatcactt gaacctggga ggcggaggtt gcggtgagcc gagatcgtgc 26280cactggattc cagcctgggt gacagagcaa gactctgtct caaaaaaaaa aaaaagaaaa 26340gaaaagaaaa gaaaagaaaa agaaacccag atgttctgtg attatcctct gaaaatcgtg 26400gtgcttaaaa aatcacggca agcatcttcc gccttgaaga gtaacagtaa tagtgacagt 26460cattagcccg acatgttgta gttgatgatc aggttcatct tcgttggctc attggaatct 26520taccaataat aaaccagtat ttgggtactc ttaccctgca acagatgggg aacaccaggc 26580caaaaggtta agcaggaatg gagaggcgtg ggaactcagg gcttcatcct cttctcgtcc 26640agggcagcac ccttcaccag gcatctccca tgtccagctc caaataagac acaggtcctc 26700gctgtcccct gtacttcctt ctagaacagc acgtggtagt ccagcagctc agataccccc 26760agtttcagca agattttggg gtttactgat tttaggtgaa ggaaaggatt ctcaatgaaa 26820acactttcat ttaaaaacag gaaatcatac atcataactc ctcctttgcc gggagagaga 26880tttttttctg catcacaatt gcaaatgcct tgcttatctt ttttttttct ttttttcccc 26940ctaatggagt cttagagcat tcctctgcgt gacataggga catggttgca ccttatctga 27000gggtcaggat ggagcagatt ggtgtgtggg tcccaggtgc cttccctgtg agggagggaa 27060gctttgctgc ttctgctcag cggctgccct ggtgatcaat tctggctaag aggaaagtac 27120attccataac atttctggtg acttgctgat catgtgatgt tgtcataata gacattgatc 27180attttatgac ccagactgat ccaatgtaaa ttcattctaa catggtgggt gctgtttcag 27240caatgagttg ttggagacct gggactcaaa ggacatctgg tccttgcctt agccagctgt 27300caaaggggtt ggaccaatag cgtttggggc cccttcagct ctcatgttga gtgaatctgt 27360gactctagga tgttgggagt gggagatggg ccaagatttg ggaccagact gaatccctgc 27420cgtcttctcc tccttctcat atttctcttc ctttcttttt tctgtttggt ctccatagtt 27480taaagtattt ctattagtca ctcaattcag ccgcattttg acttaaccag ccttgagaga 27540cctgctctaa gagccttcaa actaagttcc caacatcttt taaacttcgc tttgtccttc 27600acttctcagc tcaaatgcct gttctttgga gaagtctctc tgattgtctt atacaacagg 27660gattgaaaca ctttttgcaa aggcaagact gcaaatattg tagggcttgc aggccacagg 27720gtttctgttg caatgactga actctgccct tgtaacacac aaaagcagcg ccagacaata 27780aggaatgggc atggccgtgt tctaataaag ctttgtttac aaaaacaagc tgcggctggc 27840ttggcccgcg ggccatcatc cgccacctcc tgccaaggga gcagccctcc tctgccactg 27900ctgtgtgtgc ccgtaccttg ccatgtcctt cttcctaatg cttaagcaag aacactgtat 27960tttttgtaga ttttgtatct atcgtctgtc acccccacca taatggacgc tccacaagtg 28020cagggtcttt atcttgtctc ctcttgctgc ggagcttcca gaataggctt gttgtatagt 28080agacatgcag taaataatta ctgagtgaga aaacaaatgt tagaaacata acctgcccgt 28140cagctgggct ttccttgctt tggggtgcat ttgtccatgt tacatttagc ttctattttc 28200ctccttttgt tgtgattctc tctttgtgtt cacgtgctag ttggcatctg gggcttggca 28260tgcatgcctt tggtgatgcc cccttgagaa gccccgggcc tcctgtgtgg ctgttgatgg 28320tggtggtgag ggggatttct tgtccccaat gcatgtggcc cttgctcctt tcacagtggg 28380actgaaacac agattgactt gatttcagtc tcctgtggga gatttgttct tctgaggggt 28440gtctaggcaa aacagttgcc tttgatcctg ccctgatttg gtgctgtctc agtgctggga 28500agctttgcat tttctaggcc tggaactctg ggtcccattc acacttctca ctggagggat 28560gaaagcctcc ttcttgggcc ctcgtggcca tcccagctgg ccgctccagc tgttgttatt 28620aataaatgcc agtggtgtcc tctcaactcc agtggagagg ttgtgtgggt tggatccttg 28680tgggtccaat cccgtgctgc atatctggga actgtgacct tgtttgacat ggccatgtgt 28740ctgccaagca ggatggggtc ggtggcggga agcacagaga accatgagac ccacgccttt 28800tctctagggt cttggtgtct ccgtggggag atgggatgca ttcatgagaa tatggtgcat 28860cccatgagct ggcaaagtgg gcaggtgtat ggtggagggg gagggagcca gtgctcttcc 28920gcccaggggg actgatgcaa ggtgactccc atcacggagg accctgcttg atttgggtct 28980tggagggtgg atgacaggga agagagggag gaaggactgt ccctacagaa gaagcctggg 29040acatgtgtgg tgggtgttgg tgagcagggt gggactcagc tttgtcgtgt ggagtcgggg 29100gcacacagca cccagcggct tcatttcagg acccctgtaa gtgcaacgga gactgccttg 29160cagctgtctg ggcattttgc tctttttgtt tttggtgtta aggcctctag gcttttatca 29220ttttgggaaa acagcaggtc tcagctcacc cagcacagaa ttaggtctct aaaccggcaa 29280agtactgatc tgaacatgag agctcatgaa aactctgggt ggcaaaaaat cttgtagagg 29340gatcccttag ttttccttga gtttgagaca tttcatttta agaagcttgg ccttggggca 29400acagaccaag tctagcacaa ataataaaga gtgtatgtgt gtgtggcgca cagtcacgtt 29460tcacgtacaa atgtgtaaaa ctcggcattc ttacaaataa aacataccac aataacagat 29520ttttgcaggt attacagtgc aagcacgctt tggcaatcaa tttaaacatt ttcttgtgga 29580atattcttcc atgctatggt ttggtttgtt tctaaagccc cataggatgt ggaaactaag 29640tcaggacaga ttttctgggg gtatcctctg gttgctcttc cagatacttc ctgacttcct 29700ggggcttggg gagaggtgga tgcctgctgg ggccatttgc agagcaagaa cagcttctcc 29760tgggaataag caggcctcca ctgacctttg ttggacgttc tctcctctcc ctccttccca 29820tctgtctccg tcttctctct cccacctttc tgcagagcca ccaaccaaat accaaatctc 29880tcaaccagaa gtgtacgtgg ctgcgccagg ggagtcgcta gaggtgcgct gcctgttgaa 29940agatgccgcc gtgatcagtt ggactaagga tggggtgcac ttggggccca acaataggac 30000agtgcttatt ggggagtact tgcagataaa gggcgccacg cctagagact ccggcctcta 30060tgcttgtact gccagtagga ctgtagacag tgaaacttgg tacttcatgg tgaatgtcac 30120aggtgagttg gcccgccagc actatgctct ctcttctctg tagccattac atttttttgg 30180ccaagtgaaa aggtagtgag atctctaatt gtaattggat gccaggcata cagcttcata 30240gtttttgaaa ttcttctttg ggacctggtg caccagaaag gccgatcatt aagaatgata 30300gaattcttgt gcacaaagta acatttttct taagatagta cgcttttatt taagtaaata 30360catgcttttt tttttttttt tttgtaccac tgacatctct ggcatttaga atatagggtt 30420gaaatttgga tactcaagat ttctgattca tttattagag tttgagtttc tctccatgat 30480ttccttctat gcagtgagcg ggacagaaca ggcccccttt gtggccgagt ttaaagttct 30540gctttcagaa tgttagttga cgatgagaag ggccacacag ggactgagtt ttgttaggga 30600tcaatttctt tcttcaagga gaccccgcat actgaaaggt taatgttgga aaaagagtct 30660ttgggtgcta cacaattggt aaatttgtca ggggcttgaa tactgtttga agcttgaaat 30720ccagttctca tatatccaat tttatagcct gtttaaatag cgtgaaagca gaaaacattg 30780agaatcataa catagaccaa ctgtcatcat ggagggaaaa tttaagccat taaaactatc 30840ttaactgaaa acaatcccag gctctttgcg agggttcctg ggttgttgac tttgctatgg 30900agaaggtctc agttgtagat aattgcaacc tttttgcttt gcaaaaaaca catccatgga 30960atatgttctt ttgcatacag atgccatctc atccggagat gatgaggatg acaccgatgg 31020tgcggaagat tttgtcagtg agaacagtaa caacaagagt aagtaactgc ccggctccga 31080tggtccccga gagaggagca tggagggaag ttctgcctgt cacctgtctt cttgtcgact 31140cttctgcgcc atgctgtgtc ccgcggccct tgcctttccc cgctgtgtct actttcctga 31200ctttcaaacc tgagaataaa ccagtgttgc tgcacagcct tctctatcgt ttgtcctttc 31260ttctcgtgtc actggtcatt cgtttttcaa agcagttact acttttcttt ccttgatttt 31320cccttttccc tttgacttct ccctattcag agacataaga atagtagaac catgtaacat 31380cttggttttc cttgtaatca gtgattgtgc ttggtttaat ccagtggtgt gtgactgggg 31440caattgccta ttcttgctct cccggcacat tgggcaatat ttggttgtca caacagaggg 31500agggggtgct ggtggccttt agctggggag gggccaggga tgcagagcac agccccataa 31560caaagaatta tctggtccaa gatgtcaatc atccctaggg tgagaaaccc ggcctcctac 31620aacacacacc tcatgctgag tgaaaatgaa ggacgtgtgc cttactttgt agaccacgat 31680tgaaaaggga gccaagggtg gcttgcttaa tgagggccat gaacactgag cgctaacatg 31740ggagaggcca tttacttgcg aggaagaaca tggcgtagcc tcttggagct gggcaacctg 31800ggtttgaatc ttgctccaca acttctgagt tgctcatttc acttctgtgc cttagtttct 31860ttataaaatg ggagtaataa taataatact attttctggg gttgttatga ggattacatg 31920agttcctagt tgtatagtgc tcagaagagt ggttgcttgc aaatgtttat tcaatacaca 31980aaatacaata tagcattact tgcattttcc aatgacttgg ctagaatgtt cctaaagtgt 32040ttgtacatag ggtattggga ttctctgctt acatggtata ttctacattt ttcttaaaag 32100gattttagtc aatttggtac atttaaacaa ggcctaagta atataccaca cccggctaat 32160ttatgtatat atatatattt tttcccgaga tgcagtcttg ttctgtcacc caggctggag 32220tgcagtggcg ccatcttggc tcactgcaac ctgcacctcc caggttcaag caattctcct 32280gcctcagcct cccgagtaac taggactaca ggggcctgcc actacacctg gctaattttt 32340gtatttttag tagagatggg gtttcaccat gttggctagg ctggtctcga actcttgacc 32400ttgtgattca cctgcctcgg cctcccaaag tgctgagact acaggcgtca gccaccgtgc 32460ccagcctaat ttatgtattt ttagtagaga cagggtttta ccatgttggc caggctggtc 32520ttgaactcct gacctcaagt gatccacccg cctcagcctc ctgaagtgct gggattacag 32580gtgtgagcca ctgcgcctgg caatacttta ttttttcgag cagtttcagg tccacagcaa 32640aataaagagg aaggaacaaa gatttcccat ataatcttcc ccaacacatg catagcctgt 32700cctgttatca acatccccac cagaatggta catctgttcc agttgatgaa cctgcactgc 32760catcattatc acccaaagtg tgtggttgac tttagggtat gtaatcatac agtgtgtagc 32820ctttacagat tggcttcttt gacttagtaa gatgcatgta agtttctctc atgtcttttc 32880atggcttgat gggtcatttg tttgtagcac tgagtattcc attgtttgta tgtatcaaag 32940tttatttacc cgtttaccta ctaaaagata tctcggctgg gcaccgttgc tcacgcctgt 33000aatcctagca ctttgtgagg ccgaggcggg tggatcactt gggaacaaaa gttcgagacc 33060agcttggcca acatggcaaa atccctgtct ctgctaaaaa tacataggtt agccaagtgt 33120agtggtgcat gcctgtaatc ccagctactc gggaggctga ggcatgagaa tcacttgaac 33180ccaggatgcg gaggttgcag tgagtcgaga tcacaccact gcactccctc ctgcctggg 33239 4170 PRT Homo sapiens 4 Met Gly Leu Thr Ser Thr Trp Arg Tyr Gly Arg GlyPro Gly Ile Gly 1 5 10 15 Thr Val Thr Met Val Ser Trp Gly Arg Phe IleCys Leu Val Val Val 20 25 30 Thr Met Ala Thr Leu Ser Leu Ala Arg Pro SerPhe Ser Leu Val Glu 35 40 45 Asp Thr Thr Leu Glu Pro Glu Glu Pro Pro ThrLys Tyr Gln Ile Ser 50 55 60 Gln Pro Glu Val Tyr Val Ala Ala Pro Gly GluSer Leu Glu Val Arg 65 70 75 80 Cys Leu Leu Lys Asp Ala Ala Val Ile SerTrp Thr Lys Asp Gly Val 85 90 95 His Leu Gly Pro Asn Asn Arg Thr Val LeuIle Gly Glu Tyr Leu Gln 100 105 110 Ile Lys Gly Ala Thr Pro Arg Asp SerGly Leu Tyr Ala Cys Thr Ala 115 120 125 Ser Arg Thr Val Asp Ser Glu ThrTrp Tyr Phe Met Val Asn Val Thr 130 135 140 Asp Ala Ile Ser Ser Gly AspAsp Glu Asp Asp Thr Asp Gly Ala Glu 145 150 155 160 Asp Phe Val Ser GluAsn Ser Asn Asn Lys 165 170

That which is claimed is:
 1. An isolated peptide consisting of an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS: 1 or 3; and (d) a fragment of an amino acidsequence shown in SEQ ID NO:2, wherein said fragment comprises at least10 contiguous amino acids.
 2. An isolated peptide comprising an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequenceshown in SEQ ID NO:2, wherein said fragment comprises at least 10contiguous amino acids.
 3. An isolated antibody that selectively bindsto a peptide of claim
 2. 4. An isolated nucleic acid molecule consistingof a nucleotide sequence selected from the group consisting of: (a) anucleotide sequence that encodes an amino acid sequence shown in SEQ IDNO:2; (b) a nucleotide sequence that encodes of an allelic variant of anamino acid sequence shown in SEQ ID NO:2, wherein said nucleotidesequence hybridizes under stringent conditions to the opposite strand ofa nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a nucleotidesequence that encodes an ortholog of an amino acid sequence shown in SEQID NO:2, wherein said nucleotide sequence hybridizes under stringentconditions to the opposite strand of a nucleic acid molecule shown inSEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment ofan amino acid sequence shown in SEQ ID NO:2, wherein said fragmentcomprises at least 10 contiguous amino acids; and (e) a nucleotidesequence that is the complement of a nucleotide sequence of (a)-(d). 5.An isolated nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequence thatencodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotidesequence that encodes of an allelic variant of an amino acid sequenceshown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; (c) a nucleotide sequence that encodes anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3;(d) a nucleotide sequence that encodes a fragment of an amino acidsequence shown in SEQ ID NO:2, wherein said fragment comprises at least10 contiguous amino acids; and (e) a nucleotide sequence that is thecomplement of a nucleotide sequence of (a)-(d).
 6. A gene chipcomprising a nucleic acid molecule of claim
 5. 7. A transgenic non-humananimal comprising a nucleic acid molecule of claim
 5. 8. A nucleic acidvector comprising a nucleic acid molecule of claim
 5. 9. A host cellcontaining the vector of claim
 8. 10. A method for producing any of thepeptides of claim 1 comprising introducing a nucleotide sequenceencoding any of the amino acid sequences in (a)-(d) into a host cell,and culturing the host cell under conditions in which the peptides areexpressed from the nucleotide sequence.
 11. A method for producing anyof the peptides of claim 2 comprising introducing a nucleotide sequenceencoding any of the amino acid sequences in (a)-(d) into a host cell,and culturing the host cell under conditions in which the peptides areexpressed from the nucleotide sequence.
 12. A method for detecting thepresence of any of the peptides of claim 2 in a sample, said methodcomprising contacting said sample with a detection agent thatspecifically allows detection of the presence of the peptide in thesample and then detecting the presence of the peptide.
 13. A method fordetecting the presence of a nucleic acid molecule of claim 5 in asample, said method comprising contacting the sample with anoligonucleotide that hybridizes to said nucleic acid molecule understringent conditions and determining whether the oligonucleotide bindsto said nucleic acid molecule in the sample.
 14. A method foridentifying a modulator of a peptide of claim 2, said method comprisingcontacting said peptide with an agent and determining if said agent hasmodulated the function or activity of said peptide.
 15. The method ofclaim 14, wherein said agent is administered to a host cell comprisingan expression vector that expresses said peptide.
 16. A method foridentifying an agent that binds to any of the peptides of claim 2, saidmethod comprising contacting the peptide with an agent and assaying thecontacted mixture to determine whether a complex is formed with theagent bound to the peptide.
 17. A pharmaceutical composition comprisingan agent identified by the method of claim 16 and a pharmaceuticallyacceptable carrier therefor.
 18. A method for treating a disease orcondition mediated by a human secreted protein, said method comprisingadministering to a patient a pharmaceutically effective amount of anagent identified by the method of claim
 16. 19. A method for identifyinga modulator of the expression of a peptide of claim 2, said methodcomprising contacting a cell expressing said peptide with an agent, anddetermining if said agent has modulated the expression of said peptide.20. An isolated human secreted peptide having an amino acid sequencethat shares at least 70% homology with an amino acid sequence shown inSEQ ID NO:2.
 21. A peptide according to claim 20 that shares at least 90percent homology with an amino acid sequence shown in SEQ ID NO:2. 22.An isolated nucleic acid molecule encoding a human secreted peptide,said nucleic acid molecule sharing at least 80 percent homology with anucleic acid molecule shown in SEQ ID NOS:1 or
 3. 23. A nucleic acidmolecule according to claim 22 that shares at least 90 percent homologywith a nucleic acid molecule shown in SEQ ID NOS:1 or 3.